Novel mutations of PKD1 gene in Chinese patients with autosomal dominant polycystic kidney disease
Lan Ding1,3,
Sizhong Zhang1,3,,
Weimin Qiu1,3,
Cuiying Xiao1,3,
Shaoqing Wu1,
Ge Zhang1,3,
Lu Cheng1 and
Sixiao Zhang2
1 Department of Medical Genetics,
2 Department of Urology, West China Hospital, West China Medical Center, Sichuan University and
3 Key Laboratory of Morbid Genomics and Forensic Medicine, Sichuan Province, Chengdu 610041, People's Republic of China
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Abstract
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Background. Autosomal dominant polycystic kidney disease (ADPKD) is a common disease in China. The major gene responsible for ADPKD, PKD1, has been fully characterized and shown to encode an integral membrane protein, polycystin 1, which is thought to be involved in cellcell and cellmatrix interaction. Until now, 82 mutations of PKD1 gene have been reported in European, American, and Asian populations. However, there has been no report on mutations of the PKD1 gene in a Chinese population.
Methods. Eighty Chinese patients in 60 families with ADPKD were screened for mutations in the 3' region of the PKD1 gene using polymerase chain reactionsingle-strand conformation polymorphism (PCRSSCP) and DNA-sequencing techniques.
Results. Three mutations were found. The first mutation is a 12593delA frameshift mutation in exon 45, and the polycystin change is 4129WfsX4197, 107 amino acids shorter than the normal polycystin (4302aa). The second mutation is a 12470InsA frameshift mutation in exon 45, producing 4088DfsX4156, and the predicted protein is 148 amino acids shorter than the normal. The third one is a 11151C
T transition in exon 37 converting Pro3648 to Leu. In addition, nine DNA variants, including IVS44delG, were identified.
Conclusions. Three mutations in Chinese ADPKD patients are described and all of them are de novo mutations. Data obtained from mutation analysis also suggests that the mutation rate of the 3' single-copy region of PKD1 in Chinese ADPKD patients is very low, and there are no mutation hot spots in the PKD1 gene. Mutations found in Chinese ADPKD patients, including nucleotide substitution and minor frameshift, are similar to the findings reported by other researchers. Many mutations of the PKD1 gene probably exist in the duplicated region, promoter region, and the introns of PKD1.
Keywords: ADPKD1; gene mutation; PKD1; SSCP
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Introduction
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Autosomal dominant polycystic kidney disease (ADPKD) is a single-gene disorder, affecting approximately 1/1000 individuals in Caucasians. It is also a common disease in China, although an estimation of its prevalence is not available. ADPKD is characterized by progressive enlargement of cysts in the kidney, often leading to end-stage renal disease (ESRD) in adult life. Extra-renal abnormalities include hepatic cysts, cerebral berry aneurysms, and cardiac valve abnormalities [1,2].
ADPKD is caused by mutations in at least two different genes. PKD1, covering about 52 kb of genomic DNA on chromosome 16p13.3, consists of 46 exons and produces a 14.1 kb transcript [3,4]. Its translation product, polycystin-1, is an integral membrane glycoprotein with a large extracellular region and many transmembrane domains [5,6]. PKD2, located on chromosome 4q21-23, encodes a 5.4-kb transcript and forms a 968 amino acid product, polycystin 2 [7]. The latter is also an integral membrane protein similar to the
1 subunit of voltage-activated Ca2+ and Na+ channels [8,9]. Both polycystins interact through their C-terminal cytoplasmic tails, which suggests that PKD1 and PKD2 may function through a common signalling pathway [10,11]. The existence of a third ADPKD gene has been suggested. In Caucasians, PKD1 could explain about 85% and PKD2 approximately 1015% of the cases of ADPKD.
Detection of mutations in the PKD1 gene may provide an insight into the disease mechanism including identification of the functional critical areas of the polycystin. However, mutation detection in the PKD1 gene is complicated because besides the 3.5-kb single-copy region the other part of the gene is reiterated on the same chromosome. Until now, 82 mutations of PKD1 have been reported in different populations [1224] and a complete list of published PKD1 mutations can be found at the Cardiff Human Gene Mutation Database (http://archive.uwcm.ac.uk/uwcm/mg/search/120293.html). These mutations are nucleotide substitutions (missense/nonsense mutation), splicing mutations, deletions, and insertions. Most of them were reported in Caucasian populations, and 19 were found in Thai and Korean populations in Asia [25,26]. However, there has been no report on mutations of PKD1 gene in China.
Here we report the study of the mutation of the 3' single-copy region of the PKD1 gene in 80 patients from 60 Chinese families using single-strand conformation polymorphism (SSCP) and DNA-sequencing techniques.
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Subjects and methods
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Subjects
Eighty ADPKD patients in 60 ADPKD families from the West China Hospital, West China Medical Center of Sichuan University were included in this study. Almost all probands are individuals who accepted surgical operations or dialysis therapies in this hospital. The diagnoses of ADPKD were established by a nephrologist or urologist using imaging criteria proposed by Ravine et al. [27]. The presence of at least two cysts in each kidney of a person by ultrasonography and/or computed tomography was required. The majority of families were too small to establish a linkage to either PKD1 or PKD2. In eight families a lod score >1.0 was found with at least one of the following markers: CW2, CW4, SM6 and KG8, 3' HVR, and 24-1 [2830]. Samples from 60 healthy Chinese blood donors served as normal controls.
Polymerase chain reaction (PCR) amplification and SSCP analysis
DNA was extracted from peripheral blood leucocytes with phenolchloroform or saturated salt method. The sequences of primers used for PCR amplification were shown [12]. PCR was performed on a PE 9600 thermocycler. For SSCP analysis, 3 µl of PCR products were mixed with 3 µl of 95% formamide and 20 mM NaOH, denatured by heating to 97°C for 3 min, and loaded onto a 7% polyacrylamide/0.5x TBE gel with 2.6% cross-linking [31]. Electrophoresis was then performed at room temperature with 8001000 V for 45 h. After electrophoreris the gels were silver stained.
DNA sequencing
PCR products were cloned into either a pBluescript KS+ or a pGEM-T Easy Vector (Promega), and the obtained double-strand plasmids were sequenced using M13 Universal Primers. The PCR products were purified with PCR Cleanup Kit (Boehringer Mannheim, Mannheim, Germany) and were sequenced directly. Sequencing reactions were carried out with the Cy5 dye-terminator thermal sequenase sequencing kit (Pharmacia). Finally, products were loaded on an ALFexpress DNA sequencer (Pharmacia).
Sequence analysis
Comparison of the analysed PKD1 regions with standard sequence of PKD1 gene or its protein was performed by using the basic BLAST search and blastn in nr database of Genbank and blastx in Swissport (Internet address: http://www.ncbi.nlm.nih.gov/BLAST/).
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Results
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Deletion of an A nucleotide (in patient 52)
Four single-strand bands were found on the SSCP gel of the exon 45 for patient 52. Sequencing analysis of the PCR product revealed that there was a deletion of an A in exon 45 at nucleotide 12593 (Figure 1A
). This frameshift mutation 4129fsX4197 produces a truncated peptide, 107 amino acids shorter than the normal polycystin (4302aa).

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Fig. 1. Sequencing figures showing mutations in PKD1 gene. Arrowheads show the mutation position. (a) Abnormal sequence; (b) the normal sequence. (A) A deletion of nucleotide A in exon 45 of patient P52. (B) An insertion of nucleotide A and a G A(nt12472) substitution conserving Val4088 in exon 45 of P24. (C) Deletion of nucleotide G in intron 44 of P21.
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The patient with the deletion was first diagnosed with ADPKD when he was 36 years old. He has had bilateral renal and hepatic cysts and renal calculus. His father also suffered from ADPKD and died of ESRF when he was 63 years old. Patient 52 was diagnosed with ADPKD 8 years after his father's death, so the DNA of his father was unavailable for the present study.
Insertion of an A nucleotide (in patient 24)
Five single-strand bands were found on the SSCP gel for patient 24 when amplified with primers HH. Sequencing analysis of the PCR product cloned into the pGEM-T Easy vector showed an insertion of an extra A nucleotide in exon 45 (Figure 1B
). The 12470InsA mutation produced a frameshift mutation 4088DfsX4156, and the predicted protein would be 148 amino acids shorter than the normal polycystin (4302aa). In addition, there was a 12472G
A substitution conserving Val4088 (Figure 1B
).
Patient 24 was first diagnosed with bilateral polycystic kidneys and hepatic cysts when he was 56 years old. He developed ESRF with hypertension and diabetes 10 years later. Before this his son was also found to have haematuria and renal impairment and died aged 39 years from intracranial haemorrhage. It was very likely that his son was affected with ADPKD too. The mother of patient 24 had gastric ulcer and died of gastric haemorrhage, but it could not be confirmed if she was an ADPKD patient. Therefore, neither DNA sample from his dead mother nor from his son was available.
Missense mutation (in patient 8)
SSCP analysis using primers Gr in exon 37 of patient 8 revealed one abnormally migrating single-strand band. Then the PCR products were sequenced directly, and they showed a 11151C
T transition in exon 37, converting Pro3648 to Leu. To confirm whether this is a mutation or a polymorphism, exon 37 in 60 normal individuals or 120 normal chromosomes were analysed with primers Gr, but no similar nucleotide substitution was found.
The patient with the mutation was a 32-year-old woman. She was diagnosed as having bilateral multiple renal and hepatic cysts by ultrasonography and computed tomography. There were no other affected members in the family.
Deletion of a G in intron 44 (in patient 21)
Four single-strand bands and a slow double-strand were observed in SSCP gel of patient 21. PCR direct sequencing showed a G deletion in intron 44 of this patient. To confirm the result, PCR products were cloned into pBluescript KS+ and were sequenced, the G deletion in the patient was proved (Figure 1C
).
Other DNA variants
A few DNA variants were also detected in other ADPKD patients. For instance, band shifts were found in the SSCP gels for a patient and his affected daughter when amplified with primers JJ. For the affected father, a 12754C
T same-sense substitution conserving Ser4182 in exon 46 was detected. However, for his affected daughter, there were two substitutions in exon 45, a same-sense 12697T
G substitution conserving Ser4164 and a 12494T
C substitution conserving Leu4095. Besides, a 12472G
A same-sense substitution in exon 45 conserving Val4088 was found in two other unrelated patients. These and other sequence variants were summarized in Table 2
.
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Discussion
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Although ADPKD is a common disease in China, this is the first report of mutation analysis of the PKD1 gene in a Chinese population. Here we report three de novo mutations: a deletion and an insertion of a single nucleotide in exon 45 which both led to frameshift and truncated polypeptide chains, and a missense mutation which is a 11151C
T substitution converting Pro3648 to Leu. The two reported frameshift mutations occurred in the putative cytoplasmic C-terminus including coiled-coil domain and a heterotrimeric G-protein activation sequence [10,32]. A probable coiled-coil domain contained within the last 90 amino acids of the polycystin-1 is implicated in direct heterotypic interaction with a region of polycystin-2 from codon 872 to the C-terminal. Thus, these mutations would disrupt the interaction between polycystin-1 and polycystin-2. In addition, the two frameshift mutations, involving in the loss of the heterotrimeric G-protein activation sequence, may also affect signalling pathway.
The missense mutation 11151C
T converting Pro3648 to Leu, which was located in the cytoplasmic region between predicted transmembrane domain 7 and 8 [5], is probably disease related as this variant was not detected in controls and the amino acid proline is highly conserved in human, mouse, and fugu genes (Figure 2
). However, as proline and leucine are both non-polar amino acids, and as there is no functional assay for the PKD1 product available at present, we cannot completely rule out the possibility of a polymorphism.

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Fig. 2. Sequence alignment of human, mouse, and fugu PKD1 genes. Arrowheads show that the Proline 3648 is highly conserved in these three species.
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The patterns of mutations reported in the Chinese population is similar to that found in Caucasian and other Asian populations (http://archive.uwcm.ac.uk/uwcm/mg/search/120293.html). The mutations in the 3' single-copy region of PKD1 are rare and the mutations are mainly minor frameshift and substitute mutations (Table 1
). In addition, we have found no mutation hot spots in our patients, at least in the exons studied.
Our results are in agreement with previous studies showing that frameshift or stop mutations are not necessarily associated with a more severe phenotype. This suggests that the mutations may cause inactivation of the PKD1 product, polycystin. It has been suggested that cyst results from a two-hit mutation. According to this hypothesis, an individual inherits a mutation in one allele of PKD1 gene, and during his/her lifespan another mutation (second hit) is acquired that affects the inherited wild-type copy of the same gene [33].
Among nine DNA variants, we identified an IVS44+19delG. This DNA variant was also found in Caucasian populations. The polymorphism, 12484A
G transition conserving alanine and creating a Bsp1286I reported in the Caucasian population, was not found in our population. Indeed, when amplified PCR products were digested with Bsp1286I and then run in the non-denatured PAGE, no predicted fragments were found in healthy Chinese population and Chinese ADPKD patients. In addition, this 12484A
G transition was not found by sequencing. So some DNA variants of PKD1 gene found in Chinese are different from those found in Caucasians according to our findings (Table 2
).
When we screened the mutation of the single-copy region of PKD1 gene in Chinese patients with ADPKD, there were not as many mutations as predicted. It is reported that the mutation rate in the single-copy region of Caucasian populations is about 1015%. Taking this for our Chinese patients, six to nine mutations should have been expected. At present, only three mutations have actually been found. Therefore, more sensitive methods of detecting mutation as DGGE should be used [22]. However, it is suggested that most mutations are probably located in the duplicated area, the promoter region, or the introns of PKD1 gene.
It is also possible that hypermethylation of CpG islands in the promotor or other region of PKD1 gene could also inactivate the PKD1 gene and cause the disease.
In order to understand the molecular mechanisms of ADPKD completely, we must screen the duplicated area, the promoter region, and the introns of PKD1 in Chinese ADPKD patients, and study if there are hypermethylation of CpG islands in the promotor or other region of PKD1 [3436]. This will help us to understand the physiological role of the gene product and the disease.
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Appendix
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Family trees of three families are given in Figure 3
.
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Acknowledgments
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We thank Dr Guoyang Liu of the Medical Genetics Department at the Institute of Basic Medicine, Chinese Academy of Science for providing us 10 DNA samples of ADPKD patients. We also thank Dr Tianyong Fan and Dr Keshi Tang of the Urology Department, Dr Junming Fan of the Nephrology Department of the West China Hospital at West China Medical Center, Sichuan University, for kindly providing clinical details on some patients, and Dr Qingjie Xia for helping collect the data of some patients. We thank Dr Ying Hu of Laboratory of Population Genetics, Division of Cancer Epidemiology and Genetics, National Cancer Institute, National Institute of Health (NIH) for providing data analysis. The research was supported by the National Natural Science Foundation of China (No. 39770347).
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Notes
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Correspondence and offprint requests to: Prof. Sizhong Zhang, Deparment of Medical Genetics, West China Medical Center, Sichuan University, Chengdu 610041, The People's Republic of China. Email: szzhang{at}mcwcums.com 
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Received for publication: 18.11.00
Revision received 11. 9.01.