Putative mutation of PKD1 gene responsible for autosomal
dominant polycystic kidney disease in a Chinese family
Jing Li,1* Chaowen Yu,2* Ye Tao,1Yuan Yang,2Zhangxue Hu1and Sizhong Zhang2,3
Departments of1Nephrology and2Medical Genetics, West China Hospital, and3Division of Human Morbid Genomics, The State Key
Laboratory of Biotherapy, Sichuan University, Chengdu, Sichuan, China
PKD1 and PKD2 genes are responsible for approximately 85% and 15% of ADPKD cases, respectively. In the present study,
PKD1 and PKD2 genes were analyzed in a large Chinese family with ADPKD using denaturing high-performance liquid
in all nine affected family members, but not in any unaffected consanguineous relatives or 100 unrelated controls. These
findings suggest that the unique 4 bp insertion, c.3623-3624insGTGT, in the PKD1 gene might be the pathogenic mutation
responsible for the disease in this family.
Autosomal dominant polycystic kidney disease (ADPKD) is a common and severe renal disease. Mutations of
diagnosis, mutation screening.
autosomal dominant polycystic kidney disease, denaturing high-performance liquid chromatography, gene
Autosomal dominant polycystic kidney disease (ADPKD) is
a common inherited renal disorder that is characterized by
numerous gradually enlarged fluid-filled epithelial cysts in
bilateral kidneys and accounts for up to 10% of end-stage
renal disease.1PKD1 and PKD2 are two mapped and proven
disease-causing genes. PKD1, which is mutated in approxi-
mately 85% of ADPKD cases, encodes polycystin-1, a
receptor protein for cell–cell/matrix interactions in the regu-
lation of cell proliferation and apoptosis, whereas PKD2 is
responsible for approximately 15% of theADPKD cases and
encodes polycystin-2, a transient receptor potential (TRP)
ion channel that regulates the intracellular Ca2+concentra-
tion. Polycystin-1 interacts with polycystin-2 to form an ion
channel complex, which acts as a flow-dependent mecha-
nosensor to regulate the differentiated state of tubular epi-
Mutations of PKD1 and PKD2 are highly diversified.
Many disease-associated mutations have been recorded,
including single base substitutions, insertions, deletions and
duplications. Though a lot of them have been confirmed to
be pathogenic or not disease causing, a great number of
them still create a major difficulty or uncertainty in direct
gene diagnosis and genetic counselling, indicating the
necessity to further study the mutations and their pathoge-
nicity. Here we report a novel mutation in PKD1, which is
supposed to lead to ADPKD, in a Chinese family with
numerous patients. To provide more evidence for its patho-
genicity, the mutation has been also screened in 100 normal
controls. In connection with this, the importance of distin-
guishing the likely pathogenic mutations from the polymor-
phisms for clinical practice is also discussed.
The patient pedigree and normal controls
A large Chinese family was recruited from West China
Hospital, Sichuan University (Fig. 1). In the pedigree, 14
members were diagnosed as patients by ultrasound exami-
nation according to Ravine’s criteria6or by the descriptions
from the proband and other family living members. The
proband III5, who was severely affected by the disease, had
many cysts in the bilateral kidneys and her liver, and her
serum creatinine, uric acid and blood urea nitrogen concen-
tration were higher than other affected relatives. A total of
100 unrelated healthy volunteers were recruited as controls
after exclusion of any renal cysts by ultrasonography. Blood
samples were collected from 15 available members of the
family including III2, 3, 5, 7, 10, 11, 13, 15and IV1–7, and from all
healthy controls.The study was approved by the Institutional
Ethical Review Boards, Sichuan University, and signed
informed consent was obtained from all subjects studied.
Polymerase chain reaction amplification of
PKD1 and PKD2
Genomic DNA was extracted from peripheral blood samples
using standard phenol-chloroform procedures. Because the
Correspondence: Sizhong Zhang M.D., Department of Medical
Genetics, West China Hospital, Sichuan University, Chengdu,
Sichuan 610041, China. Email: email@example.com
*These authors contributed equally to this work.
Received 19 August 2010; accepted 19 December 2010.
International Journal of Urology (2011) 18, 240–242 doi: 10.1111/j.1442-2042.2010.02709.x
© 2011 The Japanese Urological Association
5′ region of the PKD1 gene containing exons 1–33 was
replicated three or more times in other areas on the same
chromosome, primers for five specific long-range poly-
merase chain reactions (PCR) were designed to cover this
region in order to amplify a single copy that belongs to the
PKD1 gene.7,8Then, long-range PCR products were used as
templates for 50 nested PCR reactions. Meanwhile, exons
from the unique region of the PKD1 gene and the entire
PKD2 gene were amplified from genomic DNA by 31 addi-
tional PCR reactions.9,10In total, 81 PCR products ranging
from 150 to 450 bp were separated by electrophoresis on
2.0% agarose gels to check the amplification efficiency and
were then prepared for mutation analysis.
Mutation analysis by denaturing
high-performance liquid chromatography
and DNA sequencing
DNA fragments of the PKD1 gene were analyzed by dena-
turing high-performance liquid chromatography (DHPLC)
on an automated WAVE Nucleic Acid Fragment Analysis
System (Transgenomic, Omaha, NE, USA).WAVEMAKER
4.2 software (Transgenomic, Omaha, NE, USA) was used to
determine the optimal melting temperature for the tested
fragments. Before DHPLC, the PCR products were dena-
tured at 94°C for 5 min and cooled at room temperature for
45 min. Then 6 mL of each product was injected into a
high-throughput DNASep column and eluted with a linear
acetonitrile gradient of 2% per minute at a flow of 0.9 mL/
min. The elution profiles were grouped based on the differ-
ences between normal controls and patients. Finally,
fragments with aberrant profiles were sequenced to identify
the possible variation. Because the PKD2 gene has only 15
exons, we directly sequenced the entire PKD2 gene of the
To confirm the pathogenicity or the association of the muta-
tion with the disease, careful analysis of other affected pedi-
gree members was carried out. Meanwhile, the mutation
was also checked in unaffected family members and 100
normal controls to confirm the prediction.
During the analysis of the PKD1 gene of the proband by
DHPLC, an abnormal elution peak was observed in frag-
ment 15b covering the 5′ part of exon 15 in the PKD1 gene
(Fig. 2a,c). Subsequent cloning and sequencing of the frag-
ment identified a 4 bp insertion, c.3623-3624insGTGT
(Fig. 2b,d). Segregation analysis confirmed that the patien-
ts III3, 5, 7, 10, 11, 13, 15and IV2, 5were also heterozygous for this
mutation. The unique insertion was not observed in any
non-patient family members including III1, IV1, 3, 4, 6, 7and
unrelated normal controls. This supposed disease-causing
mutation has not been reported previously in the literature,
and neither has it been recorded in The Autosomal Domi-
nant Polycystic Kidney Disease Mutation Database (PKDB)
or registered in the Human Gene Mutation Database
Analysis of the 3′sequence of the aforementioned mutated
site showed that the mutation, by causing a frameshift of the
reading frame, resulted in the premature appearance of a
new stop codon UGA, p.F1208CfsX3, which might stop the
transcription and lead to the truncation of polycystin-1. The
unique mutation was not found in any of the unaffected
consanguinious relatives and 100 unrelated controls. There-
fore, it is reasonable to speculate that truncation of the
corresponding polypeptide is probably the molecular
mechanism of ADPKD in this family. The confirmation of
the mutation as a disease-causitive also means that it might
be of some clinical diagnostic value when ADPKD is in
The present ADPKD family is relatively large and this is
rare in China, as a result of the current family planning
policy. We have noticed that the clinical symptoms and their
severity were quite diverse in different family members,
with the proband having the most severe symptoms.
Dedoussis et al. found that PKD patients with both PKD1
mutations and PKD2 variants might have a more severe
phenotype than those with only PKD1 mutations.11
However, this was not so in the present study, as we excluded
any PKD2 variants in the proband by direct sequencing, so
mal dominant polycystic kidney disease
family. The proband III5is shown by the
Pedigree of the Chinese autoso-
IV:1 IV:2 IV:3IV:4 IV:5IV:6 IV:7
III:2III:3 III:4 III:5III:6 III:7 III:8 III:9 III:10 III:11III:12III:13 III:14 III:15
II:4II:5 II:6 II:7II:8
Novel mutation in PKD
© 2011 The Japanese Urological Association
possible reasons for the more severe phenotype might be the Download full-text
living environment and other modifying factors.12
ADPKD is an important inherited nephropathy with high
prevalence, and PKD1 is a large gene with a complicated
structure, and molecular characterization and mutation
research of PKD1 really is a challenge.To date, hundreds of
different mutations have been reported, but the pathogenesis
of most of them remains unclear, which has created a major
difficulty or uncertainty for clinical use.Therefore, for clini-
cal practice, it is very important to decide whether an
any variation needs to be carefully studied, especially when
no other family member can be selected for segregation
In summary, the current study has found a unique 4 bp
insertion, c.3623-3624insGTGT, of the PKD1 gene in a
Chinese family.All evidence available suggests that it might
be the mutation responsible for causing ADPKD in that
family, and direct gene diagnosis and prenatal diagnosis in
clinical practice would be helpful.
This work was supported by the National Key Technologies
R&D Program, grant number: 2006BAI05A08, China. We
thank all patients and control subjects who participated in
1 Wilson PD. Polycystic kidney disease. N. Engl. J. Med.
2004; 350: 151–64.
2 Bycroft M, Bateman A, Clarke J et al. The structure of a
PKD domain from polycystin-1: implications for polycystic
kidney disease. EMBO J. 1999; 18: 297–305.
3 Distefano G, Boca M, Rowe I et al. Polycystin-1 regulates
extracellular signal-regulated kinase-dependent
phosphorylation of tuberin to control cell size through
mTOR and its downstream effectors S6K and 4EBP1. Mol.
Cell. Biol. 2009; 29: 2359–71.
4 Wegierski T, Steffl D, Kopp C et al. TRPP2 channels
regulate apoptosis through the Ca2+ concentration in the
endoplasmic reticulum. EMBO J. 2009; 28: 490–9.
5 Harris PC, Torres VE. Polycystic kidney disease. Annu.
Rev. Med. 2009; 60: 321–37.
6 Pei Y, Obaji J, Dupuis A et al. Unified criteria for
ultrasonographic diagnosis of ADPKD. J. Am. Soc. Nephrol.
2009; 20: 205–12.
7 Phakdeekitcharoen B, Watnick TJ, Ahn C, Whang DY,
Burkhart B, Germino GG. Thirteen novel mutations of the
replicated region of PKD1 in an Asian population. Kidney
Int. 2000; 58: 1400–12.
8 Rossetti S, Strmecki L, Gamble V et al. Mutation analysis
of the entire PKD1 gene: genetic and diagnostic
implications. Am. J. Hum. Genet. 2001; 68: 46–63.
9 Rossetti S, Chauveau D, Walker D et al. A complete
mutation screen of the ADPKD genes by DHPLC. Kidney
Int. 2002; 61: 1588–99.
10 Hayashi T, Mochizuki T, Reynolds DM, Wu G, Cai Y,
Somlo S. Characterization of the exon structure of the
polycystic kidney disease 2 gene (PKD2). Genomics 1997;
11 Dedoussis GV, Luo Y, Starremans P et al. Co-inheritance
of a PKD1 mutation and homozygous PKD2 variant: a
potential modifier in autosomal dominant polycystic kidney
disease. Eur. J. Clin. Invest. 2008; 38: 180–90.
12 Rossetti S, Burton S, Strmecki L et al. The position of the
polycystic kidney disease 1 (PKD1) gene mutation
correlates with the severity of renal disease. J. Am. Soc.
Nephrol. 2002; 13: 1230–7.
C CAGGGGCAAAGCCCGGGG GGGTTTTTT
CCAAAGCCCGG G G GT TG GG G T TTTTT
liquid chromatography. The mutation is identified by cloned
DNA sequencing. (a) A representative chromatogram with an
abnormal elution peak from a patient. (b) Insertion of a 4 bp
GTGT in exon15 of the PKD1 gene of a patient. (c) A represen-
tative chromatogram with a normal elution peak from an unaf-
fected relative. (d) Partial sequence of exon 15 in the wild-type
Chromatograms by denaturing high-performance
J LI ET AL.
© 2011 The Japanese Urological Association