Nineteen novel NPHS1 mutations in a worldwide cohort of patients with congenital nephrotic syndrome (CNS).

Abstract

Recessive mutations in the NPHS1 gene encoding nephrin account for approximately 40% of infants with congenital nephrotic syndrome (CNS). CNS is defined as steroid-resistant nephrotic syndrome (SRNS) within the first 90 days of life. Currently, more than 119 different mutations of NPHS1 have been published affecting most exons.
We here performed mutational analysis of NPHS1 in a worldwide cohort of 67 children from 62 different families with CNS.
We found bi-allelic mutations in 36 of the 62 families (58%) confirming in a worldwide cohort that about one-half of CNS is caused by NPHS1 mutations. In 26 families, mutations were homozygous, and in 10, they were compound heterozygous. In an additional nine patients from eight families, only one heterozygous mutation was detected. We detected 37 different mutations. Nineteen of the 37 were novel mutations (approximately 51.4%), including 11 missense mutations, 4 splice-site mutations, 3 nonsense mutations and 1 small deletion. In an additional patient with later manifestation, we discovered two further novel mutations, including the first one affecting a glycosylation site of nephrin.
Our data hereby expand the spectrum of known mutations by 17.6%. Surprisingly, out of the two siblings with the homozygous novel mutation L587R in NPHS1, only one developed nephrotic syndrome before the age of 90 days, while the other one did not manifest until the age of 2 years. Both siblings also unexpectedly experienced an episode of partial remission upon steroid treatment.

Full text

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Nephrol Dial Transplant (2010) 1 of 7
doi: 10.1093/ndt/gfq088
Original Article
Nineteen novel NPHS1 mutations in a worldwide cohort of patients
with congenital nephrotic syndrome (CNS)
Dominik S. Schoeb1,2, Gil Chernin1, Saskia F. Heeringa1, Verena Matejas2, Susanne Held1,
Virginia Vega-Warner1, Detlef Bockenhauer3, Christopher N. Vlangos1, Khemchand N. Moorani4,
Thomas J. Neuhaus5, Jameela A. Kari6, James MacDonald1, Pawaree Saisawat1,
Shazia Ashraf1, Bugsu Ovunc1, Martin Zenker2, Friedhelm Hildebrandt1,7
and Members of the Gesellschaft für Paediatrische Nephrologie (GPN) Study Group*
1Department of Pediatrics and Department of Human Genetics, University of Michigan, 1150 W. Medical Center Drive Drive, Ann
Arbor, MI, USA,2Institute of Human Genetics, University of Erlangen–Nuremberg, Schwabachanlage 10, Erlangen, Germany,
3Paediatric Nephrology, Great Ormond Street Hospital, Great Ormond Street, London, WC1 3JH, UK,4Department of Pediatric
Medicine, National Institute of Child Health, Rafiqi H J Road, Karachi, Pakistan,5Nephrology Unit, University Children's Hospital
Zurich, Steinwiesstrasse 75, 8032 Zurich, Switzerland,6Department of Pediatrics, King Abdul-Aziz University Hospital,
Ali Al-Murtada Road, Jeddah, Saudi Arabia and7Howard Hughes Medical Institute, Chevy Chase, Maryland
Correspondence and offprint requests to: Friedhelm Hildebrandt; E-mail: fhilde@umich.edu
*Dr. Albalwi (Riyadh, Saudi Arabia), Dr. Ariceta Iraola (Baracaldo-Bizkaia, Spain), Dr. Attrach and Dr. Shibli (Al Ain, Abu Dhabi, United Arab
Emirates), Dr. Basak (Istanbul, Turkey), Dr. Böhm (Dortmund, Germany), Dr. Bogdanovic (Belgrad, Serbia), Dr. Chadha (Richmond, USA), Dr.
Clothier and Dr. Macdonald (Birmingham, UK), Dr. Conley (Chapel Hill, USA), Dr. Cucer and Dr. Rusu (Iasi, Romania), Dr. Dixon (Cincinnati,
USA), Dr. Grillenberger (Linz, Austria), Dr. Hanan (Alexandria, Egypt), Dr. Hanevold (Augusta, USA), Dr. Hempel (Munich, Germany), Dr.
Herman (Sacramento, USA), Dr. Hodson (Sydney, Australia), Dr. Hoppe (Cologne, Germany), Dr. Keng (Penang, Malaysia), Dr. Khoury (Sydney,
Australia), Dr. Lehmann and Dr. Laube (Zürich, Switzerland), Dr. Loza (Lima, Peru), Dr. Milford (Birmingham, UK), Dr. Montoya (Munich,
Germany), Dr. Mueller (Berlin, Germany), Dr. Nayir (Istanbul, Turkey), Dr. Nissel (Rostock, Germany), Dr. Ozaltin (Ankara, Turkey), Dr. Peco-
Antic (Belgrad, Serbia), Dr. Pohl (Freiburg, Germany), Dr. Querfeld (Berlin, Germany), Dr. Rademacher (Minneapolis, USA), Dr. Serdaroglu
(Izmir, Turkey), Dr. Soliman (Cairo, Egypt), Dr. Soran (Sanliurfa, Turkey), Dr. Soylu (Izmir, Turkey)
Abstract
Background. Recessive mutations in the NPHS1 gene
encoding nephrin account for ∼40% of infants with con-
genital nephrotic syndrome (CNS). CNS is defined as ste-
roid-resistant nephrotic syndrome (SRNS) within the first
90days of life. Currently, more than 119 different mutations
of NPHS1 have been published affecting most exons.
Methods. We here performed mutational analysis of
NPHS1 in a worldwide cohort of 67 children from 62 dif-
ferent families with CNS.
Results. We found bi-allelic mutations in 36 of the 62 fam-
ilies (58%) confirming in a worldwide cohort that about
one-half of CNS is caused by NPHS1 mutations. In 26
families, mutations were homozygous, and in 10, they
were compound heterozygous. In an additional nine pa-
tients from eight families, only one heterozygous mutation
was detected. We detected 37 different mutations. Nine-
teen of the 37 were novel mutations (∼51.4%), including
11 missense mutations, 4 splice-site mutations, 3 nonsense
mutations and 1 small deletion. In an additional patient
with later manifestation, we discovered two further novel
mutations, including the first one affecting a glycosylation
site of nephrin.
Conclusions. Our data hereby expand the spectrum of
known mutations by 17.6%. Surprisingly, out of the two
siblings with the homozygous novel mutation L587R in
NPHS1, only one developed nephrotic syndrome before
the age of 90days, while the other one did not manifest
until the age of 2years. Both siblings also unexpectedly
experienced an episode of partial remission upon steroid
treatment.
Keywords: mutation analysis; nephrotic syndrome; NPHS1
Introduction
The protein nephrin [1] is an essential component of the
renal glomerular slit diaphragm [2], which is formed by
adjacent glomerular epithelial cells (podocytes). The zip-
per-like structure of the glomerular slit membrane consists
of complexes that contain the molecules neph1 and ne-
phrin, which interact between neighbouring podocyte foot
processes [3]. Nephrin contains eight immunoglobulin-
like domains, a fibronectin type III-like domain, a trans-
membranous domain and a short intracellular domain [1]
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Page 2

(Figure 1). It plays a significant role in signalling between
podocytes by interacting with molecules like CD2AP and
podocin [3]. Phosphorylated nephrin binds to Nck, an
adapter protein, hereby reorganizing the cell's actin fila-
ment network [4]. Recently, an interaction of the intracel-
lular domain of nephrin with β-arrestin was shown to
attenuate nephrin signalling [5].
Congenital nephrotic syndrome (CNS) is defined as ne-
phrotic syndrome with onset before the 90thday of postna-
tal life [6]. Recessive mutations of the nephrin encoding
gene NPHS1 were initially described in the renal histopath-
ological entity of nephrotic syndrome of the ‘Finnish type’
(CNF) [1]. However, they have more recently also been
found outside Finland [7]. Recently, mutations in nephrin
were shown to cause ∼40% of all cases of CNS [6].
Thediseaseischaracterizedbymassiveproteinuriacaused
by a disruption of the filtration barrier [8]. Due to the mas-
sive protein loss, patients often require central venous albu-
min replacement as well as parental nutrition, leading to a
high mortality from septicaemia. End-stage kidney disease
(ESKD) before the age of 2–3years and resistance to stan-
dard steroid treatment are the rules. To avoid infectious,
thromboembolicandother complications from massiveloss
ofprotein,includingimmunoglobulinsandcoagulationfac-
tors, bilateral nephrectomy, dialysis and renal transplanta-
tion at a body weight of 10kg are recommended [9].
‘Congenital nephrotic syndrome of the Finnish type’
(CNF)[10,11]isexclusivelycausedbymutationsinNPHS1
[1]. Renal histology shows microcystic dilatation of the
proximal tubules and a progressive mesangial sclerosis
[12]. Very recently, rare cases with a manifestation beyond
the age of 90days have also been published, indicating that
different mutations in NPHS1 might cause a spectrum of
clinical severity [13].
To date, 119 different mutations in NPHS1 are known.
To expand the spectrum of known mutations, we per-
formed mutational analysis of NPHS1 by direct sequenc-
ing of all exons in 67 patients from 62 different families
with CNS.
Materials and methods
Patients and data recruitment
DNA samples and clinical data of a worldwide cohort of 2 056 children
with nephrotic syndrome (NS) were ascertained between 1996 and 2008.
The diagnosis was made by paediatric nephrologists on the basis of pub-
lished criteria [14]. Nephrotic-range proteinuria was defined as protein-
uria >40mg/m2/h. After informed consent was obtained, detailed
clinical data and pedigree information were referred to us by the specia-
lists through a standardized clinical questionnaire (www.renalgenes.org)
[15]. For all the patients, we performed mutational analysis of NPHS2
encoding podocin and WT1, the most frequent monogenic causes of child-
hood NS. Human subject research was approved by the University of Mi-
chigan Institutional Review Board and the Ethics Commission of the
UniversityofFreiburg,Germany.Outofthisworldwidecohort,weselected
65 children from 62 different families who had CNS and in whom NPHS2
(podocin) and WT1 were excluded. Two patients manifested later, but their
siblings had CNS. Also, mutation analysis in phospholipase C epsilon 1
(PLCE1) was negative for the six patients with CNS who had a renal his-
tology of diffuse mesangial sclerosis (DMS) [16]. In all 67 patients, muta-
tion analysis for NPHS1 was performed by PCR with exon-flanking
primers followed by direct sequencing. When evaluating frequency of mu-
tations,werelatethemtofamiliesratherthanpatientsbecausesiblingshave
identical mutations. When evaluating clinical data, we relate them to pa-
tients because siblings might differ in clinical phenotype.
Mutation analysis
GenomicDNAwasisolatedfrombloodsamplesusingthePuregene®DNA
purification kit (Gentra, Minneapolis, MN) following the manufacturer's
guidelines. Mutation analysis was performed by direct sequencing of all
29 exons of NPHS1, all eight exons of NPHS2 and exons 8 and 9 of
WT1. WT1 analysis was limited to exons 8 and 9 because mutations of this
geneaccountingforisolatedNShaveonlybeenreportedinthesetwoexons
[17,18]. Additionally, for seven patients with a renal histology of DMS, all
exons of PLCE1 were examined by direct sequencing. Exon-flanking pri-
mersforNPHS1,PLCE1,NPHS2andWT1havebeenpublishedpreviously
[15,16,18,19]. For sequence analysis, the software Sequencher 3.8 (Gene
Codes, Ann Arbor, MI) was used. As reference for NPHS1, the published
wild-type sequence (NM_004646) was used for nucleotide and amino acid
Fig. 1. Localization of mutations in nephrin. The nephrin protein consists of eight extracellular Ig-like domains (Ig 1–8), a fibronectin type III-like
module (Ig, FN3), a transmembrane domain (Ig) and a C-terminal (C) cytoplasmic domain (curled line). The grey/white background delimits the exons
coding for the corresponding protein domains. All mutations found in this study are listed (novel mutations—white on black; known mutations—black
on white). Note that mutations were spread throughout the protein with predominance of Ig-like domain 5. The patient harbouring these mutations was
not included in the study cohort (asterisk).
2 D.S. Schoeb et al.

Page 3

Table 1. All NPHS1 mutations detected
Patient numberExon Nucleotide exchangea
Effect on coding sequence Mutation statusb
SegregationReference
Homozygous mutations
A2471
A2201
A3023
A2911
A1176
A2031
A2617
CNS11 II-1
CNS11 II-2
A2300
A1804
CNS09 II-1
CNS09 II-2
A2417
A945
A2236 II-1
A2236 II-2
A2355
CNS05
A2330
F1273
CNS03
A2088
A2538
A3075
A2553
CNS12
A2210 II-1
A2210 II-3
A2036
4
4
8
9
9
9
9
9
9
10
11
13
13
13
14
14
14
14
14
20
20
21
24
24
24
26
27
28
28
28
c.C500T
c.514_516delACC
c.C896T
c.C1019A
c.G1040A
c.A1096C
c.C1099T
c.G1134A
c.G1134A
c.C1219T
c.G1379A
c.C1672T
c.C1672T
c.A1757G
c.1759-15_1778del
c.T1760G
c.T1760G
c.T1760G
c.G1868T
c.2815+5G>A
c.2664-4_2670del
c.2927+1G>A
c.3243_3250insG
c.3243_3250insG
c.3243_3250insG
c.C3325T
c.3481+1G>T
c.C3478T
c.C3478T
c.C3478T
p.P167L
p.T172del
p.R299C
p.P340H
p.G347E
p.S366R
p.R367C
p.W378X
p.W378X
p.R407W
p.R460Q
p.R558C
p.R558C
p.R586G
Splice error
p.L587R
p.L587R
p.L587R
p.C623F
Splice error
Splice error
Splice error
p.1084fsX12
p.1084fsX12
p.1084fsX12
p.R1109X
Splice error
p.R1160X
p.R1160X
p.R1160X
Hom
Hom
Hom
Hom
Hom
Hom
Hom
Hom
Hom
Hom
Hom
Hom
Hom
Hom
Hom
Hom
Hom
Hom
Hom
Hom
Hom
Hom
Hom
Hom
Hom
Hom
Hom
Hom
Hom
Hom
p,m
nd
nd
nd
nd
nd
nd
?
?
p,m
nd
?
?
p,m
p,m
p,m
p,m
p,m
?
p,m
nd
?
nd
nd
nd
nd
?
nd
nd
nd
ps
Lenkkeri et al. 1999
ps
ps
ps
Lenkkeri et al. 1999
Lenkkeri et al. 1999
ps
ps
ps
Beltcheva et al. 2001
Beltcheva et al. 2001
Beltcheva et al. 2001
ps
ps
ps
ps
ps
Lenkkeri et al. 1999
ps
Heeringa et al. 2008
ps
Kestila et al. 1998
Kestila et al. 1998
Kestila et al. 1998
Kestila et al. 1998
Beltcheva et al. 2001
Lenkkeri et al. 1999
Lenkkeri et al. 1999
Lenkkeri et al. 1999
Compound heterozygous mutations
A23411
3
5
20
6
13
6
22
8
14
9
19
9
24
9
24
15
17
15
24
17
27
c.58+1G>T
c.C320T
c.C574T
c.T2728C
c.613_620delinsTT
c.G1715A
c.613_620delinsTT
c.G2928T
c.G886A
c.T1760G
c.T1048C
c.G2625A
c.A1096C
c.C3478T
c.C1019A
c.C3478T
c.G2043T
c.2227delC
c.C2019A
c.C3478T
c.C2227T
c.C3442T
Splice error
p.A107V
p.Q193X
p.S910P
p.T205,P206,R207>I205
p.S572N
p.T205,P206,R207>I205
p.R976S
p.W289X
p.L587R
p.S350P
p.W875X
p.S366R
p.R1160X
p.P340H
p.R1160X
c.W681C
p.R743fsX10
p.N673K
p.R1160X
p.R743C
p.Q1148X
Cpd hetm
p
nd
nd
nd
nd
m
p
?
?
?
?
nd
nd
nd
nd
p
m
p
m
nd
nd
ps
ps
ps
ps
Lenkkeri et al. 1999
Gigante et al. 2005
Lenkkeri et al. 1999
ps
ps
ps
Lenkkeri et al. 1999
ps
Lenkkeri et al. 1999
Lenkkeri et al. 1999
ps
Lenkkeri et al. 1999
ps
ps
ps
Lenkkeri et al. 1999
Lenkkeri et al. 1999
Beltcheva et al. 2001
A2535b
Cpd het
A1943 Cpd het
A2616Cpd het
CNS01 Cpd het
CNS08 Cpd het
A2030 Cpd het
A3083 Cpd het
A2475A1981 Cpd het
Cpd het
A2249 Cpd het
Single mutations only
A2618
A2380
A1869
A1517
A2250
A2062
A2320 II-1
A2320 II-2
3
10
11
14
17
19
23
23
c.C320T
c.G1223A
c.G1379A
c.G1868T
c.C2227T
c.G2625A
c.C3418T
c.C3418T
c.A107V
c.R408Q
c.R460Q
c.C623F
c.R743C
c.W875X
c.R1140C
c.R1140C
het sm
het sm
het sm
het sm
het sm
het sm
het sm
het sm
m
nd
nd
nd
nd
p
nd
nd
ps
Lenkkeri et al. 1999
Beltcheva et al. 2001
Lenkkeri et al. 1999
Lenkkeri et al. 1999
ps
Lenkkeri et al. 1999
Lenkkeri et al. 1999
As relevant wild-type gene sequence, the published reference sequence of NPHS1 was used (NM_004646). het, heterozygous; Hom, homozygous
mutation; cpd het, compound heterozygous mutation; sm, single mutation only detected; p, paternal; m, maternal; nd, not done; ?, information not
available for this patient; ps, novel mutation detected in the present study.
aAll mutations were absent from 93 healthy controls.
bPatient was not included in the study.
Novel NPHS1 mutations in nephrotic syndrome3

Page 4

numbering. For all detected mutations and variants, both strands were se-
quenced. Whenever possible, segregation was confirmed by direct se-
quencing of the parental samples. For novel mutations, their absence
from 93 healthy control individuals was confirmed by direct sequencing.
Homozygosity mapping
Genome-wide homozygosity mapping for 12 families with CNS was per-
formed and evaluated as described previously [20]. Single-nucleotide
polymorphism (SNP) arrays (GeneChip®) from Affymetrix, Inc. with a
resolution of 250K (Human Mapping 250K Styl Array) were used.
Samples were processed, hybridized and scanned using the manufac-
turer's standard methods at the University of Michigan Core Facility
(www.michiganmicroarray.com). Using the software Allegro [21] and
ALOHOMORA [22], non-parametric likelihood ratio Z-scores (ZLRs)
were calculated using one marker every 100 000 markers. Allele frequen-
cies for Caucasians as specified by Affymetrix®, a disease allele frequency
of 0.001, and a standard pedigree structure assuming first-cousin marriage
for parents of affected individuals were used. ZLRs were calculated under
threedifferentconditions,i.e.forminorallelefrequenciesof>0.2,>0.3and
>0.4, and a non-existent sibling was included to enable non-parametric Al-
legro runs. If a peak was constantlyexceeding thevalue of 2.0 in two out of
the three conditions, we referred to it as a ‘consistent ZLR peak’ (cZLR)
and expected it to harbour the homozygous mutation of the recessive
disease gene [20]. The ZLRs were plotted against genetic distance
across the entire human genome using the Gnuplot software (http://
www.gnuplot.info) (Supplementary Figure 1; see online supplementary
material for a colour version of this figure). In this way, the maxima of
ZLR scores represent segments of homozygosity by descent.
Results and discussion
Clinical characteristics and ethnicity of patients
In this study, 67 patients (32 females, 34 males and 1 with
unknown gender) from 62 different familieswith CNS were
included.AllpatientswereexaminedforNPHS1mutations.
Renal biopsy was performed in 24 patients, showing 10
with a pattern congruent to NS ‘Finnish type’, 8 patients
with DMS, 3 with minimal change nephrotic syndrome
(MCNS), 2 with focal segmental glomerulosclerosis
(FSGS), 1 with membranoproliferative glomerulonephritis
(MPGN)and1withend-stagenephrosclerosis(Supplemen-
taryTable2).Altogether,21differentethnicitieswererepre-
sented within the cohort; among these, the largest groups
were of Turkish (15%), Arabic (15%), European (13%)
and Caucasian (9%) descent (Supplementary Table 2).
Consanguinity was reported in 20 families (Supplementary
Table 1). In three cases, a nephrectomy had been per-
formed, and 10 patients had received a renal transplant
(Supplementary Table 2).
Modality of treatment and response
Because traditionally CNS is considered treatment refrac-
tory, steroid treatment was only reported as attempted in 14
(20.8%) cases. Eleven of these patients showed no re-
sponse (steroid-resistant nephrotic syndrome, SRNS), as
expected for CNS, while three (A2236 II-1/II-2, A2380)
did partially respond. In four cases, cyclosporin A was ap-
plied in addition to steroids, while one patient was treated
with cyclosporin A only (Supplementary Table 2). In none
of these subjects was any response recorded (Supplemen-
tary Table 2, Supplementary Table 4).
‘Antiproteinuric therapy’ with angiotensin-converting
enzyme (ACE) inhibitors or indomethacin was attempted
in 34 patients. In nine patients, exclusively ACE inhibitors
were applied, of which two (22.2%) showed a partial remis-
sion (A1804, A2475). Partial remission is hereby under-
stood as the permanent disappearance of oedema, an
increase in the serum albumin concentration to >35g/L
and the persistence of proteinuria of >4mg/m2/h [23]. In
both patients, mutation analysis had revealed disease-caus-
ing mutation in NPHS1. Surprisingly, A2475 showed com-
pound heterozygosity for a missense mutation and a
deletion. A combined therapy with indomethacin and
ACE inhibitors was administered to 26 patients of which
eight (30%) showed partial remission, while in 17 cases,
no effect was observed. Of the eight patients showing a par-
tial remission, a disease-causing mutation in NPHS1 was
found in six patients. Of these six patients, missense muta-
tions were disease causing in five cases. Unexpectedly,
however, patient A2201 also showed partial remission, al-
though a homozygous deletion was detected in this patient.
He was treated with captopril for 40months followed by
10months of treatment with losartan. Indomethacin was
applied for 3months but did not show any benefit. These
data show that ‘antiproteinuric’ therapy has a beneficial
effect on 20–30% of patients with CNS and should not
only be considered for patients with missense mutations
but might also be positive for patients with a more severe
type of mutation (Supplementary Table 2, Supplementary
Table 4).
For 19 out of the 67 patients, no pharmacological treat-
ment was reported.
NPHS1 mutations
In 67 children from 62 different families, mutation analysis
by direct sequencing of all NPHS1 exons was performed.
In 36 families, causative mutations in NPHS1 were de-
tected on both alleles. We hereby confirm the results of
former studies [6,19] showing that approximately one-half
of CNS cases are caused by recessive mutations in NPHS1
(Table 1). In CNS, it has been shown that ∼85% of the
cases are explained by mutations in four genes. The distri-
bution among these four genes is: NPHS1 39.8%, NPHS2
39.8%, WT1 2.2% and LAMB2 4.4% [6]. Twenty-six of the
36 families showed homozygous mutations, and another
10 families had compound heterozygous mutations. In
seven families, only one heterozygous NPHS1 mutation
was detected (Table 1). As a reason for this relatively high
number of patients with disease-causing mutations, only on
one allele, one might speculate that deletions/duplications
of whole exons as well as intronic mutations and mutations
in the promoter region can explain these cases. A direct
sequencing approach might not have been able to detect
these mutations.
Out ofthe37different disease-causing NPHS1 mutations
detected, 19 mutations were novel, consisting of 11 mis-
sense mutations, 4 splice-site mutations, 3 nonsense muta-
tions and 1 deletion. They were found in exons 1, 3, 4, 8, 9,
10, 13, 14, 15, 17, 19 and 21 (Table 1 and Figure 1, Supple-
mentary Figure 1; see online supplementary material for a
colour version of this figure). The 18 previously published
mutations consisted of 10 missense mutations, 3 nonsense
mutations, 1 insertion, 1 deletion, 2 splice-site mutations
4D.S. Schoeb et al.

Page 5

and 1 insertion/deletion. They were found in exons 4, 6,
9, 10, 11, 13, 14, 17, 20, 22, 24, 26 and 27 (Table 1,
Figure 1). None of the patients had the Finmajoror the
Finminormutation. The mutations were broadly distributed
over the nephrin protein, affecting all domains. The most
frequently affected domains were immunoglobulin (Ig)-like
domain 5 and Ig-like domain 3 (Figure 1).
Genotype/phenotype correlations
While the CNS classical histology of ‘Finnish type’rapidly
progresses into ESKD and shows no response to treatment,
several cases of patients with NPHS1 mutations have been
reported, whose histological phenotype was not as severe
and who sometimes even showed a response to treatment
[19,24].
From the 12 patients in our study, in whom renal biopsy
was performed and whose disease was explained by two
recessive mutations in NPHS1, eight patients showed NS
‘Finnish type’, three showed MCNS, one showed DMS
and one showed FSGS (Supplementary Table 3). These da-
ta confirm the previous finding that renal pathology does
not exclusively appear as NS ‘Finnish type’ in CNS caused
by recessive NPHS1 mutations. A higher frequency of mu-
tations in a certain ethnicity was not observed.
Out of the seven patients in our cohort with a biopsy of
DMS, only one had disease-causing NPHS1 mutations
(A2911II-1).Thispatientshowedanovelhomozygousmis-
sense mutation (c.1019C>A, p.P340H) (Table 1). The pa-
tient was steroid resistant. Manifestation was at birth, and
ESKD developed at the age of 4months (Supplementary
Table 1). Another patient (A3083 II-1) showed the same
mutation heterozygously together with a heterozygous
known nonsense mutation [c.3478 C>T (h), p.R1160X]
(Table 1). He manifested at the age of 2months and also
showed steroid resistance. Biopsy was not performed, and
ESKD was not reported by the age of 6months (Supple-
mentary Table 1). DMS is seen in patients with mutations
in PLCE1, WT1 and LAMB2. As WT1 and PLCE1 yielded
no mutations in the remaining DMS patients, we speculate
that they may have mutations in LAMB2 or PLCE1.
One patient (A2616 II-1) with a biopsy of FSGS
showed compound heterozygosity for a known deletion
(c.613_620delinsTT; p.T205, P206, R207>I205) and a
novel missense mutation [c.2928G>T (h), p.R977S]
(Table 1). The age of disease onset was 2months, and
steroid treatment was not attempted. The patient devel-
oped ESKD by the age of 10years and was transplanted
(Supplementary Table 1).
In two patients with a homozygous NPHS1 mutation
(CNS03, CNS11 II-1), a renal biopsy of MCNS was re-
ported. Patient CNS03 II-1 had a truncating mutation
[c.1134G>A (H), p.W378X], while patient CNS11 II-1 had
asplice-siteerror (c.2927 +1G>A, splice error) (Table 1). In
bothcases,notreatmentwasattempted.Theageofonsetfor
patient CNS03 was shortly after birth, and patient CNS11
II-1 manifested later at the age of 4months. For patient
CNS11 II-2, no biopsy was performed; both siblings were
treated with steroids for 2months but did not respond (Sup-
plementaryTable1).PatientCNS03showedsevereprogres-
sion with nephrectomy at the age of 6months and histology
of glomerulosclerosis as well as tubular atrophy, microcysts
and interstitial fibrosis. As patients with an early biopsy of-
ten show MCNS but progress rapidly, the aetiopathology of
thispatientisnotsurprising.However,patientCNS11II-1is
reportedto bestabileat theage of2.5yearsnowand, togeth-
er with the late onset, is showing a rather unusual course of
disease.Bothsiblingsalsoshowedpartialremissiontotreat-
ment with ACE inhibitors and indomethacin.
AlthoughmutationsinNPHS1werethoughttoexclusive-
lycause CNF,these results confirmformer findingsindicat-
ing that NPHS1 mutations can cause a somewhat broader
variety of histological phenotypes in nephrotic syndrome.
In a recent genetic study of patients with CNS [6], in a total
of 21 patients with two NPHS1 mutations, the histological
phenotypes were distributed as follows: ‘Finnish type’
(14%), MCNS (14%), FSGS (4.6%), DMS (3.6%), mesan-
gial proliferation (9.2%), mesangial sclerosis (3.6%) and no
finding (3.6%) [6] (Supplementary Table 3). In 10 patients,
nobiopsywasperformed.Theseresultswerealsoconfirmed
byanotherstudyofchildrenwithCNS,showingagain‘non-
Finnish type’ manifestations [19] (Supplementary Table 3).
While the CNS classical histology of ‘Finnish type’rap-
idly progresses into ESKD and shows no response to treat-
ment, several cases of patients with NPHS1 mutations have
been reported, whose histological phenotype was not as
severe and who sometimes evenshowed a response to treat-
ment [19,24]. Recently, Phillipe et al. even reported several
cases with childhood rather than congenital onset of ne-
phrotic syndrome and confirmed mutations in NPHS1
[13]. In this study, two siblings were included who had dif-
ferent age of onset. While the male sibling (A2236 II-2)
manifested as CNS by the age of 2.5months, his elder sister
(A2236II-1)stayedhealthyuntiltheageof24months.Both
showed partial remission due to steroid treatment, and in
both sibs, a novel homozygous missense mutation
(c.1760T>G, p. L587R) was detected. Partial remission is
hereby understood as the permanent disappearance of oe-
dema, an increase in the serum albumin concentration to
>35g/L and the persistence of proteinuria of >4mg/m2/h
[23]. A third patient (A2355 II-1), who was classified as
CNS and of the same ethnicity, showed the same mutation.
Interestingly, he also was not diagnosed before the age of 5
months.Steroidtreatmentforthispatientwasnotattempted.
Additionally, one patient (CNS01) showed L587R hetero-
zygouslyincombinationwithaheterozygousnonsensemu-
tation[c.886G>A(h),W289X].Thispatientshowedtheage
of onset of 2months, and no form of steroid treatment was
reported. We therefore conclude that homozygous L587R
may be a milder mutation, causing a less severe form of ne-
phrotic syndrome than other NPHS1 mutations with possi-
ble childhood onset later than 90days of life. It is, to our
knowledge, the first homozygous mutation in NPHS1 to
cause childhood onset (in A2236 II-1 and A2355 II-1),
and the findings suggest that mutation analysis should also
be sought in children who manifest after 90days of life.
Detection of NPHS1 mutations by total genome
homozygosity mapping
Mutations that are homozygous by descent have been
described as being frequent (30–80%) in paediatric dis-
Novel NPHS1 mutations in nephrotic syndrome5

Page 6

eases [20]. This can be mapped by homozygosity map-
ping. In order to investigate if homozygosity mapping is
a useful tool for screening, we performed homozygosity
mapping in a subset of this cohort of 12 patients from
12 families with different background (Supplementary
Table 1). All of them exhibited homozygous segments
by inspection of their homozygosity plots, while only
five (A2031 II-1, F1273 II-1, A1804 II-1, A2088 II-1,
A2036 II-1) revealed homozygosity at the NPHS1 locus
(Supplementary Figure 2; see online supplementary ma-
terial for a colour version of this figure). We detected
homozygous disease-causing mutations of NPHS1 in
all five patients. Of the remaining seven patients, two
(A1981 II-1, A2062 II-1) showed compound heterozy-
gous disease-causing mutations, two (A1517 II-1, A1869
II-1) showed single heterozygous mutations and three
(A1970 II-1, A1980 II-1, A2112 II-1) showed no mutations
of NPHS1 (Table 1, Supplementary Table 1).
Mutation in N-glycosylation site
In addition to the results of our systematic mutation
screening in a CNS cohort, we report on a single patient
with onset of nephrotic syndrome at the age of 9months
who was also found to be mutated in NPHS1. The patient
was not included in the study, and his clinical data is not
shown here. He was treated with steroids but did not re-
spond (SRNS). His renal biopsy showed the histological
features of IgM nephropathy.
Mutation analysis of NPHS1 in patient A2535 revealed
two novel heterozygous mutations: c.574C>T; p.Q193X
and c.2728T>C; p.S910P (Table 1, Figure 1, not included
in the examined cohort). This finding is of interest as, to
ourknowledge,S910Pisthefirstmutationdescribed,which
directly affects one of nephrin's ten known N-glycosylation
sites [25]. Substitution of the serine residue by proline is
predicted to prevent glycosylation at this site. Defects in
post-translational modification may lead to decreased
stability of impaired interaction with other molecules.
Considering the late manifestation of nephrotic syn-
drome in this patient, we speculate that the mutation
S910P has some residual protein function and might
be a ‘mild’ mutation. As it has been described recently,
a ‘mild’ mutation in combination with a ‘severe’ muta-
tion in NPHS1 may cause childhood onset of nephrotic
syndrome [13].
Adding these mutations, we here report 21 novel muta-
tions, expanding the number of published mutations in
NPHS1 by 17.6%.
Supplementary data
Supplementary data is available online at http://ndt.oxford-
journals.org.
Acknowledgements.
tribution of blood samples and clinical data. This work was supported by
a grant to F.H. from the National Institutes of Health (DK076683, RC1-
DK086542), and the Thrasher Research Fund, and by a grant to M.Z. by
the German Research Foundation (DFG; SFB 423). F.H. is an investigator
of the Howard Hughes Medical Institute, a Doris Duke Distinguished
Clinical Scientist and a Frederick G.L. Huetwell Professor.
We thank the patients and their physicians for con-
Conflict of interest statement. None declared.
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Received for publication: 3.8.09; Accepted in revised form: 02.02.10
Novel NPHS1 mutations in nephrotic syndrome7