A missense mutation in PKD1 attenuates the severity of renal disease.

Abstract

Mutations of PKD1 and PKD2 account for most cases of autosomal dominant polycystic kidney disease (ADPKD). Compared with PKD2, patients with PKD1 typically have more severe renal disease. Here, we report a follow-up study of a unique multigeneration family with bilineal ADPKD (NFL10) in which a PKD1 disease haplotype and a PKD2 (L736X) mutation co-segregated with 18 and 14 affected individuals, respectively. In our updated genotype-phenotype analysis of the family, we found that PKD1-affected individuals had uniformly mild renal disease similar to the PKD2-affected individuals. By sequencing all the exons and splice junctions of PKD1, we identified two missense mutations (Y528C and R1942H) from a PKD1-affected individual. Although both variants were predicted to be damaging to the mutant protein, only Y528C co-segregated with all of the PKD1-affected individuals in NFL10. Studies in MDCK cells stably expressing wild-type and mutant forms of PKD found that cell lines expressing the Y528C variant formed cysts in culture and displayed increased rates of growth and apoptosis. Thus, Y528C functions as a hypomorphic PKD1 allele. These findings have important implications for pathogenic mechanisms and molecular diagnostics of ADPKD.

Full text

Page 1

A missense mutation in PKD1 attenuates the
severity of renal disease
York Pei1, Zheng Lan2, Kairong Wang1, Miguel Garcia-Gonzalez2,3, Ning He1, Elizabeth Dicks4,
Patrick Parfrey4, Gregory Germino2,5and Terry Watnick2
1Division of Nephrology, University of Toronto, Toronto, Ontario, Canada;2Division of Nephrology, Johns Hopkins School of Medicine,
Baltimore, Maryland, USA;3Laboratorio de Investigacion en Nefrologia, Complexo Hospitalario Universitario de Santiago, Santiago de
Compostela, Spain;4Division of Nephrology, Memorial University, St John’s, Newfoundland, Canada and5National Institute of Diabetes
and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, USA
Mutations of PKD1 and PKD2 account for most cases of
autosomal dominant polycystic kidney disease (ADPKD).
Compared with PKD2, patients with PKD1 typically have
more severe renal disease. Here, we report a follow-up
study of a unique multigeneration family with bilineal
ADPKD (NFL10) in which a PKD1 disease haplotype and
a PKD2 (L736X) mutation co-segregated with 18 and
14 affected individuals, respectively. In our updated
genotype–phenotype analysis of the family, we found that
PKD1-affected individuals had uniformly mild renal disease
similar to the PKD2-affected individuals. By sequencing
all the exons and splice junctions of PKD1, we identified
two missense mutations (Y528C and R1942H) from a
PKD1-affected individual. Although both variants were
predicted to be damaging to the mutant protein, only Y528C
co-segregated with all of the PKD1-affected individuals in
NFL10. Studies in MDCK cells stably expressing wild-type and
mutant forms of PKD found that cell lines expressing the
Y528C variant formed cysts in culture and displayed
increased rates of growth and apoptosis. Thus, Y528C
functions as a hypomorphic PKD1 allele. These findings have
important implications for pathogenic mechanisms and
molecular diagnostics of ADPKD.
Kidney International (2012) 81, 412–417; doi:10.1038/ki.2011.370;
published online 26 October 2011
KEYWORDS: ADPKD; functional assay; hypomorphic allele; missense variant;
mutation analysis
Autosomal dominant polycystic kidney disease (ADPKD) is
the most common hereditary kidney disease worldwide and
accounts for B5% of end-stage renal disease (ESRD) in
North America.1,2It is characterized by focal and age-
dependent development of renal cysts, leading to massive
enlargement and distortion of the normal architecture of
both kidneys and, ultimately, to ESRD in a majority of
patients. Mutations of two genes, PKD1 and PKD2, were
thought to account for 85 and 15% of cases, respectively, in
linkage-characterized European populations.3However, more
recent population-based series suggest a higher prevalence
of PKD2 ranging from 26 to 36%.4Disease progression of
ADPKD is highly variable and in part due to a strong gene
locus effect.5–9Adjusted for age and gender, PKD1 patients
have larger kidneys and earlier onset of ESRD compared with
PKD2 patients (mean age at ESRD: 53.4 vs. 72.7 years,
respectively).5–8By contrast, a weak allelic effect (based
on the 50vs. 30location of the germline mutations) on renal
disease severity may be present for PKD1,7but not for
PKD2.8In addition, marked intrafamilial renal disease
variability in ADPKD suggests a modifier effect from genetic
and environmental factors.7–10
PKD1 is a large gene consisting of 46 exons with an open
reading frame of approximately 13kb and is predicted to
encode a protein of 4302 amino acids. Its entire 50region up
to exon 33 has been duplicated six times on chromosome
16p, and the presence of these highly homologous pseudo-
genes has made genetic analysis of PKD1 challenging.1,2
Recent availability of protocols for long-range and locus-
specific amplification of PKD1 has enabled complete muta-
tion screening of this complex gene.11–14By contrast, PKD2 is
a single-copy gene consisting of 15 exons with an open
reading frame of approximately 3kb and is predicted to
encode a protein of 968 amino acids.1,2Recent studies of two
large cohorts of patients with ADPKD have indicated that
PKD1 is a highly polymorphic gene.13,14On average, 10–13
polymorphic variants were identified in PKD1 compared
with B1 in PKD2. Marked allelic heterogeneity is also
evident for ADPKD, with over 400 different PKD1 and B100
different PKD2 mutations reported to date that are thought
original article
http://www.kidney-international.org
& 2012 International Society of Nephrology
Received 9 June 2011; revised 8 September 2011; accepted 13
September 2011; published online 26 October 2011
Correspondence: York Pei, Division of Nephrology, University Health
Network, 8N838, 585 University Avenue, Toronto, Ontario, Canada M5G
2N2. E-mail: york.pei@uhn.on.ca or Terry Watnick, Division of Nephrology,
Johns Hopkins School of Medicine, 855 North Wolfe Street, Rangos 551,
Baltimore, Maryland 21205, USA. E-mail: twatnick@jhmi.edu
412
Kidney International (2012) 81, 412–417

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to be definitively or highly likely pathogenic (http://pkdb.
mayo.edu/cgi-bin/mutations.cgi).2,11–15The majority of these
mutations are unique and scattered throughout both genes.
In general, protein-truncating
deletion/insertion, nonsense changes, or splice defects)
mutations, which are considered definitively pathogenic,
have been identified in only 47–63% of patients.12–14By
contrast, ‘unclassified variants’ (e.g., in-frame deletions and
missense variants resulting in non-synonymous amino-
acid substitutions) with pathogenic potential have been
detected in an additional 26–37% of patients.13–14Although
the clinical significance of the latter class of sequence variants
remains to be defined, two recent studies suggest that certain
missense PKD1 mutations might function as hypomorphic
alleles associated with mild or atypical renal disease.16,17
Here, we provide further evidence to support this emerging
concept.
In 2001, we reported a large multigeneration family
(NFL10) with bilineal ADPKD in which a PKD1 disease
haplotype and a PKD2 (L736X) mutation co-segregated with
18 and 14 affected individuals, respectively (Figure 1).18After
excluding the PKD2-affected subjects in this family, we
performed linkage analysis and obtained a highly significant
(because of frame-shift
LOD (logarithm (base 10) of odds) score of B5.0 at KG8
(an intragenic marker within PKD1), strongly suggesting that
the disease in the remaining affected members was due to
PKD1. Consistent with our linkage results, we found perfect
segregation of a putative PKD1 disease haplotype (D16S521-
HBAP1-KG8-D16S665-D16S291-D16S2618:
in the 16 affected members who did not have the PKD2
mutation. Two additional subjects with trans-heterozygous
mutations of both genes had more severe renal disease com-
pared with subjects affected with only PKD1 or PKD2 alone,
providing for the first time evidence for genetic interaction in
ADPKD. Interestingly, the renal disease of the PKD1-affected
subjects in NFL10 appeared very mild and was indistinguish-
able from those affected with PKD2.
3-4-1-7-8-5)
RESULTS
Genotype–phenotype correlation
In the current report, we have updated the most recent infor-
mation on the renal function of the affected subjects from
NFL10 with an additional follow-up of 7.5 years. Confirm-
ing our previous impression, we found that PKD1-affected
subjects continued to have mild disease that was indis-
tinguishable from that of PKD2-affected subjects (Figure 2a).
OP100
(66)
OP108
(62)
OP107
(58)
OP103
(66)
OP8
(48)
OP154
OP155
OP12
(72)
OP13
(52)
OP3
(65)
OP160
(80)
OP161
(69)
OP127
(68)
OP120
(69)
OP121
(66)
OP124
(63)
OP129
(35)
OP18
(41)
OP17
(33)
OP16
(23)
OP57
(47)
OP53
(46)
OP49
(41)
OP45
(37)
OP29
(27)
OP30
(33)
OP36
(45)
OP40
(43)
OP102
(48)
OP64
(22)
OP65
(19)
PKD1:
D16S521
HBAP1
KG8
D16S665
D16S291
D16S2618
PKD2:
D4S231
D4S1534
SPP1
D4S1563
D4S423
2
4
6
6
9
3
4
1
7
8
5
OP20
(44)
Figure 1|Disease segregation pattern in NFL10. Fourteen affected individuals carrying the PKD2 (L736X) mutation co-segregated with
the PKD2 haplotype (D4S231-D4S1534-SPP1-D4S1563-D4S423: 2-4-6-6-9; denoted by the red bar), whereas 16 affected individuals without
the PKD2 mutation co-segregated with the putative PKD1 disease haplotype (D16S521-HBAP1-KG8-D16S665-D16S291-D16S2618: 3-4-1-7-8-
5; denoted by the blue bar). Two members (OP8 and OP13) of this family affected with autosomal dominant polycystic kidney disease
carried both mutations. The number in parentheses denotes the age at the most recent clinical assessment. Double horizontal line denotes
marriage between two related individuals.
Kidney International (2012) 81, 412–417
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Y Pei et al.: Hypomorphic PKD1 allele with mild renal disease
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Indeed, the linear regression analysis showed that the slope of
estimated glomerular filtration rate (eGFR) by age did not
differ among the affected subjects with the two gene types
(?1.61 (95% confidence interval: ?1.25 to ?1.97) for PKD1
vs. ?1.94 (95% confidence interval: ?1.45 to ?2.44) for
PKD2). Moreover, compared with a large cohort of PKD1
patients, those individuals with Y528C clearly had better
preserved renal function. Four PKD1-affected members were
over 60 years of age at their last clinical follow-up and none
had developed ESRD. Renal imaging studies were available
for two of these older PKD1-affected subjects. A computed
tomography scan performed on OP103 at age 60 years
showed normal-sized kidneys (right 6.9?6.5?12cm; and
left 8.1?7.6?11.1cm) with innumerable subcentimeter-
sized small cysts located bilaterally (Figure 2b). Similarly, an
ultrasound performed on OP100 at age 59 years showed
normal-sized kidneys (right 11cm and left 12cm in length)
with numerous small renal cysts located bilaterally and three
liver cysts. These extremely mild findings of renal cystic
disease are highly atypical of PKD1.
Identification of a pathogenic PKD1 mutation
To identify the pathogenic PKD1 mutation in NFL10,
bidirectional sequencing of all the exons and splice junctions
of PKD1 was performed in the genomic DNA of OP100,
and 26 sequence variants were detected (Supplementary
Table S1 online). Most of the sequence variants were known
or predicted polymorphisms, except for two novel missense
variants, Y528C and R1942H, both of which were predicted
to be damaging by PolyPhen (http://genetics.bwh.harvard.
edu/pph/). The PSIC profile score difference was 2.4 for
Y528C and1.8for R1942H.
(c.1583A4G) co-segregated with all 18 PKD1-affected
individuals (including the two trans-heterozygotes, OP8
and OP13). The Y528C variant affects an amino-acid residue
in the C-type lectin domain of polycystin-1 (PC1) that is
highly conserved across multiple species (Figure 3) and
moderately conserved across non-PC1 proteins with the same
domain (Supplementary Figure S1 online).
However, onlyY528C
In vitro analysis of Y528C
To establish the pathogenicity of Y528C, we performed
in vitro functional studies in stable MDCK cell lines
expressing tetracycline-inducible wild-type and mutant
(Y528C) forms of PC1. We found that both wild-type and
mutant PC1 were equally expressed and that the Y528C
mutation did not disrupt the normal cleavage of PC1 at the
GPS site (Supplementary Figure S2 online).14We have
previously established that MDCK cells form cysts when
grown in three-dimensional collagen cultures. Expression of
PC1 in these cells is sufficient to induce tubulogenesis along
with reduced growth rates and resistance to apoptosis.19We
found that MDCK cell lines expressing Y528C continued to
form cysts in culture, similar to what has been demonstrated
for a fully penetrant mutant control.20In addition, Y528C-
expressing cell lines demonstrated increased rates of growth
and apoptosis, similar to what was observed in the empty
vector control (Figure 4). One of the limitations of this
in vitro assay is its inability to discriminate between complete
and partial phenotypes. However, taken together with the
uniformly mild renal phenotype observed in individuals
harboring Y528C, the above data strongly suggest that this
variant is the pathogenic PKD1 mutation in NFL10 and that
it functions as a hypomorphic allele in vivo.
DISCUSSION
Three conclusions can be drawn from our study on NFL10.
First, the observation that PKD1-affected individuals con-
sistently displayed attenuated renal disease argues in favor of
an allelic effect associated with the Y528C mutation, rather
than a modifier effect.9,10Second, our study is consistent with
140
120
100
80
60
40
20
0
0
b
a
eGFR (ml/min per 1.73 m2)
20 4060
Age (years)
80
PKD1 Y528C
PKD2 mutation
Both mutations
PKD1 Ref.cohort
Figure 2|Genotype–phenotype correlation in NFL10. (a) Renal
disease severity in subjects affected with PKD1 (Y528C) and PKD2
was very similar in this pedigree. Linear regression analysis
showed that the slope of estimated glomerular filtration rate
(eGFR) by age did not differ between affected subjects with the
two gene types (PKD1: ?1.61 (95% confidence interval (CI): ?1.25
to ?1.97); PKD2: ?1.94 (95% CI: ?1.45 to ?2.44)). However,
two subjects affected with both PKD1 and PKD2 had more
severe renal disease compared with subjects affected with only
PKD1 or PKD2 alone. For comparison, the mean eGFR (95% CI)
from a large cohort of PKD1 patients is also presented according
to the age strata of 20–29 (n¼44), 30–39 (n¼92), 40–49 (n¼132),
50–59 (n¼79), and 60–69 (n¼30) at ages 25, 35, 45, 55, and 65
years, respectively. The CI was not shown in the last age group,
as the eGFR was not normally distributed. (b) The computed
tomography scan performed on OP103 at age 60 years showed
only mildly enlarged kidneys with numerous small cysts in the left
kidney and multiple small cysts in the right kidney. A large left
peripelvic renal cyst and a single liver cyst were also noted.
414
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Page 4

the recent concept that a critical cellular polycystin threshold
is involved in cystogenesis. Previous studies have provided
strong evidence in support of a cellular recessive model
in which homozygous inactivation of PKD1 or PKD2 by
germline and somatic mutations could trigger cystogenesis
in ADPKD.21–23However, recent studies on homozygous
Pkd1 knockout mice with aberrant mRNA splicing indicate
that complete biallelic inactivation may not be absolutely
required, as renal cystogenesis can be triggered by normal
PC1 levels below a 10–20% threshold.24,25Within the ‘two-hit
model’ framework, data from our current and two recent
studies16,17are consistent with the notion of a critical cellular
polycystin threshold for cystogenesis.24,25Third, two recent
molecular diagnostic surveys of patients with ADPKD have
identified protein-truncating mutations in only 47–63% of
the cases, with 26–37% of additional cases reported to have
‘unclassified variants’ (in-frame deletions and non-synon-
ymous sequence variants) with pathogenic potential.13,14
From the latter patient cohort, it is likely that more cases of
hypomorphic PKD1 alleles will be increasingly recognized.
An outstanding question of major clinical importance is
what proportion of these ‘unclassified variants’ function as
hypomorphic alleles and therefore might be associated with
mild renal disease. This can only be answered by systematic
genetic–phenotype analyses involving a large number of cases.
In the meantime, unambiguous demonstration of hypo-
morphic PKD1 alleles from recent clinical studies implies that
mild renal disease is no longer restricted to patients with
PKD2 (refs 5 and 8) and further extends the spectrum of
renal disease variability in ADPKD.
MATERIALS AND METHODS
Study subjects and clinical assessment
We have previously ascertained the pedigree structure of NFL10
consisting of at least 130 members over six generations. Forty-four
at-risk individuals and four spouses who gave informed consent were
studied. All the at-risk individuals and spouses were screened with at
least one abdominal ultrasound. All the scans were performed under
the supervision of one ultrasonographer and interpreted by a
radiologist (Dr Benvon Cramer, Department of Radiology, Memorial
University, St John’s, Newfoundland) without any knowledge of the
clinical status of the subjects using the Ravine criteria.26The research
protocols used for this study were approved by the Human Subject
Review Committees of the University of Toronto and Memorial
University in Newfoundland. In this update, we repeated the
serum creatinine measurements in 30 of 32 affected subjects and
reassessed their renal function as eGFR using the MDRD formula.27
Two affected subjects with ESRD were assigned a default eGFR of
10ml/min. For comparison purpose, we used the data from a large
cohort of PKD1 patients (excluding NFL10) that we had studied
previously28to generate mean eGFR (95% confidence interval) by
each decade plotted at the midpoints of the following age groups:
20–29 (n¼44), 30–39 (n¼92), 40–49 (n¼132), 50–59 (n¼79), and
60–69 (n¼30). We did not present any confidence interval for the last
age group, as the data were not normally distributed.
Mutation screening
Mutation screening of PKD1 and PKD2 was performed for
OP100 using a commercial diagnostic service (Athena Diagnostics,
Worcester,MA;http://www.athenadiagnostics.com/).13
DNA was used as a template for specific long-range PCR ampli-
fication of eight segments encompassing the entire PKD1 duplicated
region. The long-range PCR products served as templates for 43
Genomic
b
a
Normal
control
OP100
Human
Dog
Rat
Mouse
Opossum
Chicken
Frog
Pufferfish
C-type lectin domain
Y528
(nt: 1583A>G; p: Y528C)
CCGCACAGCT
A
ACCCGGGGTT
Figure 3|Co-segregation of Y528C with PKD1 disease haplotype in NFL10. (a) A highly conserved PKD1 missense (nt: A1583G;
p: Y528C) mutation co-segregated with all 18 affected subjects with the putative PKD1 disease haplotype. (b) Y528C affects an
amino-acid residue in the C-type lectin domain of polycystin-1, which is highly conserved across multiple species including the
pufferfish, fugu.
Kidney International (2012) 81, 412–417
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Y Pei et al.: Hypomorphic PKD1 allele with mild renal disease
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nested PCRs, whereas the unique region of PKD1 and the entire
PKD2 were amplified from genomic DNA in 28 additional PCRs. All
71 PCR products were bidirectionally sequenced, including the
coding regions and exon–intron splice junctions of both genes. To
confirm the segregation of disease-associated variants, long-range and
nested PCR products for exons 7 (for Y528C) and 15 (for R1942H)
were amplified from genomic DNA using published primers and
conditions.14The nested PCR products were then sequenced.
Predicting deleterious missense mutations
We used the software PolyPhen to evaluate the functional impact of
a number of unclassified missense variants that alter a single amino-
acid residue. Upon entry of the protein ID, and the wild-type and
mutant amino-acid variants, PolyPhen conducts a comprehensive
search to identify all the homologous protein sequences. On the
basis of alignment of these homologous protein sequences, PolyPhen
computes profile scores for both the allelic variants.28Profile scores
are logarithmic ratios of the likelihood of a given amino acid
occurring at a particular site to the likelihood of this amino acid
occurring at any site (background frequency). A variant is predicted
to be damaging if the absolute difference between the profile scores
of two amino-acid variants is 41.7.29
In vitro functional analysis
The wild-type PKD1 construct was derived from pCI-PKD1-Flag.19
The Y528C mutant was generated from this base construct using
site-directed mutagenesis (Stratagene, La Jolla, CA). Stable MDCK
cell lines expressing flag-tagged wild-type or mutant (Y528C) PC1
were generated using an Flp-In tetracycline inducible system
(Invitrogen, Carlsbad, CA). A vector-only cell line was used as a
control. PC1 expression was induced with tetracycline (TET) and
the cells were cultured with selection agents hydromycin (100mg/ml)
and blasticidin (5mg/ml). The tubulogenesis assay was performed
using the methods reported by Boletta et al.19Cells forming cysts or
tubules were assayed 2 weeks after culture and tabulated for three
independent clonal isolates. After the cells were serum starved for
72h, apoptosis was assessed by terminal deoxytransferase uridine
triphosphate nick end labeling staining and cell proliferation was
cd
Proliferation rate
BrdU assay
(%)
40
a
pCDNA5
PKD1
Y528C
TET
−+
Apoptosis assay
80
(%)
Apoptosis positive
FLP PKD1Y528C
60
40
20
0
30
20
10
0
FLPPKD1Y528C
b
pCDNA5
TET
PKD1
Y528C
−+−+−+
Cyst
Tubule
123456
979919139999
31
81
87
1
1
100
80
60
40
20
0
Figure 4|In vitro functional analysis of stable MDCK cell lines expressing wild-type and mutant Y528C forms of PKD1.
(a) Representative example of the tubulogenesis assay in the absence (?) or presence (þ) of tetracycline (TET). Almost 100% of the
vector control cells form cysts, whereas PKD1-expressing cells form tubules regardless of induction with tetracycline. On the basis of
western analysis this is explained by leaky polycystin-1 expression, which occurs even in the absence of tetracycline. The Y528C cell line
exhibits ‘mutant’ behavior and forms cysts. The arrow indicates a tubule formed by a PKD1-expressing cell. (b) The percentage of cells
forming cysts or tubules was assayed 2 weeks after culture and was tabulated for three independent clonal isolates. (c) Apoptosis assay
by terminal deoxytransferase uridine triphosphate nick end labeling staining. The vector control (FLP) consistently exhibits an apoptotic
rate of B45%. The apoptotic rate drops markedly to less than 10% in PKD1-expressing cell lines. The Y528C-expressing cell line shows
a relatively high apoptotic rate that is not statistically different from vector control. (d) Cell proliferation by 5-bromodeoxyuridine (BrdU)
incorporation. PKD1-expressing cells had a lower rate of proliferation when compared with the vector control. Growth rates for Y528C
resembled the control.
416
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Y Pei et al.: Hypomorphic PKD1 allele with mild renal disease

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assessed by 5-bromodeoxyuridine incorporation.19All the assays
were performed in triplicate. Antibodies used for immunodetection
of PC1 have been previously described.20
Statistical analysis
Linear regression analysis on the rate of loss of eGFR on the basis
of age in subjects affected with PKD1 or PKD2 was performed
using the equation fitting suite of the software SlideWrite Plus
version 7.01 (Advanced Graphics Software, Santa Fe, CA).
DISCLOSURE
All the authors declared no competing interests.
ACKNOWLEDGMENTS
We are indebted to all the participating members of the NFL10
family. This work was supported by grants from the Canadian
Institutes of Health Research (CIHR; MOP77806) to YP, from the
National Institutes of Health (RO1DK0617 and RO1GM73704) to TW
and (DK48006 and NIDDK Intramural Research Program) to GG, and
by the CIHR Distinguished Scientist Award to PP. These studies
utilized Resources provided by the NIDDK-sponsored Johns Hopkins
PKD Research and Clinical Core Center (P30 DK090868).
SUPPLEMENTARY MATERIAL
Table S1. PKD1 and PKD2 gene variants identified from OP100.
Figure S1. Alignment and comparison of 44 non-redundant full-
length sequences from proteins with C-type lectin domain by
Pfam (http://pfam.sanger.ac.uk/) showing moderate conservation
(score 4/10) at residue Y528 (boxed in red).
Figure S2. Y528C did not alter polycystin cleavage at the GPS site.
Supplementary material is linked to the online version of the paper at
http://www.nature.com/ki
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Y Pei et al.: Hypomorphic PKD1 allele with mild renal disease
original article