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Novel and de novo PKD1 mutations identified by multiple restriction fragment-single strand conformation polymorphism (MRF-SSCP)
© Thongnoppakhun et al; licensee BioMed Central Ltd. 2004
Received: 20 November 2003
Accepted: 03 February 2004
Published: 03 February 2004
We have previously developed a long RT-PCR method for selective amplification of full-length PKD1 transcripts (13.6 kb) and a long-range PCR for amplification in the reiterated region (18 kb) covering exons 14 and 34 of the PKD1 gene. These have provided us with an opportunity to study PKD1 mutations especially in its reiterated region which is difficult to examine. In this report, we have further developed the method of multiple restriction fragment-single strand conformation polymorphism (MRF-SSCP) for analysis of PKD1 mutations in the patients with autosomal dominant polycystic kidney disease (ADPKD). Novel and de novo PKD1 mutations are identified and reported.
Full-length PKD1 cDNA isolated from the patients with ADPKD was fractionated into nine overlapping segments by nested-PCR. Each segment was digested with sets of combined restriction endonucleases before the SSCP analysis. The fragments with aberrant migration were mapped, isolated, and sequenced. The presence of mutation was confirmed by the long-range genomic DNA amplification in the PKD1 region, sequencing, direct mutation detection, and segregation analysis in the affected family.
Five PKD1 mutations identified are two frameshift mutations caused by two di-nucleotide (c. 5225_5226delAG and c.9451_9452delAT) deletions, a nonsense (Q1828X, c.5693C>T) mutation, a splicing defect attributable to 31 nucleotide deletion (g.33184_33214del31), and an in-frame deletion (L3287del, c.10070_10072delCTC). All mutations occurred within the reiterated region of the gene involving exons 15, 26, 15, 19 and 29, respectively. Three mutations (one frameshift, splicing defect, and in-frame deletion) are novel and two (one frameshift and nonsense) known. In addition, two mutations (nonsense and splicing defect) are possibly de novo.
The MRF-SSCP method has been developed to analyze PCR products generated by the long RT-PCR and nested-PCR technique for screening PKD1 mutations in the full-length cDNA. Five mutations identified were all in the reiterated region of this gene, three of which were novel. The presence of de novo PKD1 mutations indicates that this gene is prone to mutations.
Autosomal dominant polycystic kidney disease (ADPKD; MIM# 173900) is a potentially fatal disease and one of the most common inherited disorders in humans, affecting approximately 1 per 1,000 individuals. The characteristic abnormality is the presence of multiple fluid-filled cysts in both kidneys causing defective functions, ultimately leading to end stage renal disease (ESRD) . Liver cysts and other extra-renal manifestations such as cerebral aneurysm and mitral valve defect, can also be observed . Two known genes are responsible for this disease: PKD1 (MIM# 601313) at 16p13.3 [3–6] and PKD2 (MIM# 173910) at 4q21-23 . The presence of the third locus responsible for this disease is still controversial [8, 9]. A majority of cases (85%) is caused by mutations in PKD1.
PKD1 gene spans >50 kb of genomic DNA, containing 46 exons and transcribing ~14-kb mRNA [3–6] that encodes a large membrane-associated glycoprotein, polycystin-1, comprising 4,302 amino acids. The protein possibly participates in the signal-transduction pathway by coordinating with polycystin-2, the product of PKD2, in mediating mechanosensory calcium mobilization by function as flow-sensitive mechanosensors in the primary cilium of renal epithelium . A failure of fluid-flow sensation of the cells may disturb tissue morphogenesis, triggering abnormal cell proliferation and cyst formation. Renal cysts are likely to develop after a second-hit or somatic mutation, which inactivates the inherited normal allele of the same locus, or occasionally an allele of another counterpart locus (i.e.PKD2 or vice versa), giving rise to a trans-heterozygous event .
To date, at least 250 unique PKD1 mutations have been identified as listed in the Human Gene Mutation Database (HGMD, http://uwcmml1s.uwcm.ac.uk/uwcm/mg/search/120293.html)  and also reported more recently [13–15]. Three-fourths of mutations in this gene result in truncated protein products, suggesting its inactivation or loss-of-function mechanism. Most previous studies have found unclear genotype-phenotype correlations for PKD1 mutations. However, a recent report has shown the significant correlation between the position of mutation and disease severity (more severe in more 5' mutation), raising the questions whether PKD1 mutations simply inactivate all products of the gene . Mutation databases from worldwide population and additional experiments would be useful to unravel the precise mechanism by which PKD1 mutations cause diverse phenotypes.
Analysis of PKD1 mutations is labor-intensive, time-consuming, and expensive because of its large size, the presence of a very high GC content, and most severely the presence of more than six homologous genes, which are highly homologous to the 5' region (exons 1–33) of PKD1 [4, 17]. The latter has made mutation detection in this gene much more difficult. The methods successfully developed for mutation analysis of this gene are based on discrimination of PKD1 from the non-functional homologous genes, by enrichment of PKD1 sequence while suppressing the homologous ones to undetectable levels. PKD1 mutations occur throughout the gene with apparently no mutational "hot-spots", rendering characterization of all disease causing PKD1 mutations in various ethnic groups essential for comprehension of molecular mechanism of the disease and for providing diagnostic genetic testing. Our group aim to fulfill PKD1-mutation database of Thai patients and have previously developed a long RT-PCR technique for isolation of the entire coding sequence of PKD1 (~13 kb) for the analysis of its mutations without interference from the homologous sequences . The analysis of PKD1 transcript provides an opportunity for ex vivo study of splicing defects prior to identification of causative mutations. In addition, a long-range PCR (LR-PCR) for amplification of PKD1-specific genomic sequence with the length of about 18 kb in the reiterated region covering exons 14–33 has also been established for further analysis of PKD1 . Using these techniques, we have previously identified and reported two mutations causing splicing defects of PKD1 transcripts in Thai families with ADPKD [19, 20].
To increase efficiency of screening and identification for other types of mutations in the reiterated region of PKD1, we have further developed a multiple restriction fragment-single strand conformation polymorphism (MRF-SSCP) method , in addition to the long RT-PCR and LR-PCR techniques. Since the SSCP method is more efficient for screening mutation in a short fragment (less than 300 bp), we used different sets of restriction endonucleases for digestions of each PKD1-cDNA segment so that the entire segment was efficiently screened. We have applied this procedure for screening and identifying PKD1 mutations in Thai patients with ADPKD. Here, we report three novel and two recurrent mutations of PKD1 identified in Thai patients; two mutations are possibly de novo.
ADPKD families and linkage study
Ten unrelated Thai families with ADPKD attending our clinics at Divisions of Medical Genetics and Nephrology, Department of Medicine, Faculty of Medicine Siriraj Hospital, Bangkok, were included in this study. ADPKD was diagnosed according to established clinical and genetic criteria . Research investigation was conducted in accordance with the Faculty of Medicine Siriraj Hospital Ethics Committee's guidelines and in accordance with the Helsinki Declaration. Blood samples were collected after informed consent for RNA and DNA preparations. Linkage analysis was performed by using 5 polymorphic DNA markers on chromosome 16p including D16S85 (3' HVR), SM7, 16AC2.5, SM6, and KG8 as previously described .
Full-length PKD1cDNA amplification by long RT-PCR
PCR primers and conditions for amplifications of PKD1 cDNA and DNA
Primer Sequence (5'->3')
Location in PKD1Transcript
PCR Product Size (bp)
Annealing Temperature (°C)
Mg2+ Conc. (mM)
Genomic DNA amplification
Screening of PKD1mutation by MRF-SSCP
MRF-SSCP for mutation analysis of the full-length PKD1 cDNA
Size (bp) before
Restriction fragment length
(bp) in MRF-SSCP (5' > 3')
Pvu II+Xcm I+Ava II
209, 318, 99, 249, 294, 385
Afl III+Sac I+Ava II
209, 282, 384, 238, 312, 129
Pvu II+Hinf I
429, 93, 366, 277, 295, 218
Sma I+Bgl I
108, 245, 292, 605, 123, 306
Bgl I+Hinf I
233, 81, 424, 198, 325, 202, 112
429, 215, 187, 243, 501
Bgl I+Hha I
131, 159, 96, 380, 108, 476, 123
178, 252, 475, 204, 253, 111
Sau3A I+Bgl I
127, 387, 377, 188, 226, 169, 189
Ava II+Bgl I
116, 324, 423, 216, 141, 85, 358
Ava II+Bgl I
173, 307, 294, 402, 187, 143
BstU I+BstE II
313, 344, 265, 190, 70, 239, 85
BstU I+Hinf I
112, 287, 424, 389, 201, 105
Ava II+Hinf I
197, 202, 398, 315, 192, 109, 105
Pvu II+Pst I+BstE II
337, 199, 233, 505, 218, 185
Taq I+Bgl I
190, 141, 222, 344, 474, 98, 208
190, 351, 81, 180, 453, 135, 12, 248
Taq I+Bgl I
288, 318, 197, 529, 84, 234
The digestion of nested-PCR fragments was performed in a final volume of 25 μl, containing 10 μl of DNA, 2.5 μl of 10× buffer, and 2–5 units of enzymes (e.g. Ava II + Hinf I or Bgl I + Hha I), in the presence or absence of BSA. The reaction mixture was incubated either at 37, 60 or 65°C according to the enzymes used and following the manufacturer's recommendations. Five μl of the digested-PCR product were mixed with 5 μl of 5× TBE and 10 μl of SSCP-loading dye (95% formamide, 0.05% bromophenol blue, 0.05% xylene cyanol, 20 mM EDTA and 10 mM NaOH). The total mixture was heat-denatured at 95°C for 10 min before loading onto the vertical electrophoresis set (size: 17 × 15 × 0.8 cm3) of 10% polyacrylamide gel containing 5% glycerol. The electrophoresis was run at a constant current (20 mA) and room temperature for 4–7 h; the running time was longer for a larger size of the digested fragments. DNA fragments on the gel were stained with silver nitrate . After that, the gel was placed between two sheets of cellophane, left at room temperature to dry, and kept as a record.
The fragment with mobility shift was amplified with a newly designed pair of primers. The PCR product was purified and subjected to a direct sequencing using ABI PRISM™ BigDye™ Terminator Cycle Sequencing Ready Reaction Kit and ABI PRISM 310 Automated DNA Sequencer (PE Applied Biosystems, Foster City, CA, USA).
Genomic DNA analysis
A long-range PCR (LR-PCR) was performed to amplify genomic DNA in the PKD1 region covering the nucleotide positions 26340–44438 (18,099 bp). The amplified product was then used as template for nested-PCRs (Figure 1B). The primers used for nested-PCRs are listed in Table 1. The methods for LR-PCR and nested-PCR have been previously described . Briefly, the reaction was performed in a total volume of 25-μl containing 2 μl of the diluted LR-PCR product, 400 nM of each primer, 200 μM dNTP mixture, 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.25 unit AmpliTaqGold™ (PE Applied Biosystems) or IMMOLASE™ DNA polymerase (Bioline USA Inc.), 10% DMSO, and an optimal concentration of MgCl2. The nested-PCR product was purified for DNA sequencing to confirm the nucleotide change observed in the PKD1 cDNA.
Once the mutation was identified, a simple detection method was developed for direct mutation and segregation analysis in the affected family. In addition, the mutation was examined in 56 DNA samples from unrelated normal individuals (112 chromosomes). The disease-causing mutation should not be observed in these normal DNA samples.
Of ten families studied, ADPKD was found to be PKD1-linked or probable PKD1-linked by linkage (haplotype) analysis using 5 polymorphic markers in the PKD1 region on chromosome 16p in eight families. The results of haplotype analysis in two families were inconclusive because of small number of family members.
PKD1 mutations in Thai families identified by our group
RNA Splicing Defect (experimentally determined)
Effect on Polycystin-1 (theoretically deduced)
Del the first 74 nt of exon 14
FS at 1055 (PKD IV-terminal deletion)
Thongnoppakhun et al., 2000 
FS at 1672 (PKD XI – terminal deletion)
Stop at 1828 (PKD XIII-terminal deletion)
This study, Rossetti et al., 2002 
g.33184_33214del31 (31bpE19I19-2 to E19I19+29 or c.7913_7914+29del31)
Exon 19 skipping
FS at 2498 (REJ module-terminal deletion)
This study (Novel)
FS at 3082 (TM 1-terminal deletion)
This study (Novel)
L3287del (c.10070_10072delCTC or g.41669_41671delCTC)
Inframe deletion of L3287 (TM 2)
This study (Novel)
Exon 43 skipping
Inframe deletion of 3904–4001 (TM 7–9)
Rungroj et al., 2001 
Direct detection of this 31-bp deletion in genomic DNA samples of the family members could be conducted by LR-PCR of PKD1 and nested PCR using the pair of SI6.1F/SI6.1B primers. The result showed that only the proband carried this novel deletion; none of other six family members including the patient's mother had the mutation; thus, it is likely to be a de novo PKD1 mutation (Figure 2C).
Mutation segregation study by Pvu II digestion in the PK031 family demonstrated that three affected members (I-1, II-1, and II-2) carried this mutation while one non-affected member (I-2) did not (Figure 3C and 3D).
The proband (II-I) of the family PK069 attended our renal clinics with the problem of end-stage chronic kidney disease at the age of 42 years. She was found to have polycystic kidney disease and since then she commenced on hemodialysis. Three of her siblings (II-2, II-3, and II-5) also had the disease but with normal renal function. Her father was still healthy at the age of 81 years. Her mother died of cervical cancer at the age of 38 years. PKD might be inherited from her mother. Mobility shifts were found in the MRF-SSCP analysis of SI 7 fragment from PKD1 cDNA of the proband (II-1) (Figure 4D). The final sequence analysis in both cDNA and PKD1-specific DNA indicated a deletion of AT at codons 3080 and 3081 (c.9451_9452delAT or g.39376_39377delAT), resulting in a frameshift translation starting from codon 3082 (Figure 4E). This mutation abolishes an AfI III restriction site. Analysis of the mutation by Afl III digestion in the available members of family PK069 demonstrated that all four affected persons (II-1, II-2, II-3, and II-5) carried the mutation while two unaffected members (I-1 and II-4) did not (Figure 4F).
Sequence alignments of polycystin-1 among distant species from human through worm (Caenorhabditis elegans) reveal that L3287 is highly conserved (Figure 5C). In addition, screening in our control set of 112 normal chromosomes did not show the L3287del in PKD1. The L3287del mutation could directly be detected by the heteroduplex analysis of the amplified PKD1-specific fragment. The presence of L3287del mutation in five affected members (II-1, II-2, III-2, III-4, and III-6) but its absence in eight unaffected members of PK002 family were demonstrated (Figure 5D).
Single nucleotide polymorphisms
PKD1 polymorphisms detected in Thai population
Amino acid change
Presence in HGsa
BsmA I (+)
Hph I (+)
Bsp1286 I (-)
Pvu II (-)
We have developed a long RT-PCR method for amplification and isolation of the entire PKD1-coding sequence from its RNA transcripts prepared from peripheral blood lymphocytes, avoiding the interference from transcripts of PKD1-homologous genes . This amplified full-length PKD1 cDNA is a useful material for mutation analysis. Since the amplified product was originated from PKD1 mRNA, abnormal transcripts resulted from mutations causing defective RNA splicing can directly be analyzed without the requirement for additional expression experiment. In addition, this amplified product can also be used for analysis of other PKD1 mutations. An LR-PCR for genomic-DNA amplification of 18 kb covering exons 13–34 region of PKD1 has also been developed for confirmatory mutation analysis in genomic DNA . In our previous works, we have demonstrated the use of long RT-PCR and nested-PCR procedure and LR-PCR method for characterization of two PKD1 mutations causing splicing defects in two Thai families [19, 20]. The advantage of simultaneous characterization of abnormal RNA splicing with the mutation has also been demonstrated in a recent work by Burtey et al.  who developed a method for PKD1 mutation screening by RT-PCR and reported three splicing defects out of six mutations identified. Unlike our RT-PCR and nested-PCR method that require only 10 PCR reactions per one sample, the recently reported method  needs as many as 57 PCR reactions per one sample.
Another advantage of the method that we developed is that it uses reagents and equipments available in general molecular genetic laboratory. In addition, the mutation screening method can be modified to increase efficiency and to be suitable for each laboratory. However, several disadvantages are present in analysis of PKD1 transcript including difficulty in sample handling and storage because of its unstable nature making it unpopular for routine service laboratory, complicated experimental procedure in cDNA synthesis, and presence of false-positive results due to contamination of atypical illegitimate products which sometimes puzzle interpretation of result and make it necessary to analyze of PKD1-genomic DNA for confirmation of mutation.
In this study, we have further applied the developed long RT-PCR followed by nested PCR and long-range genomic-DNA amplification for analysis of mutation in the reiterated region of PKD1 in Thai families with ADPKD. The MRF-SSCP technique has been developed and preliminarily used for screening other types of mutations. This method is able to screen mutation in a long cDNA fragment (>1 kb) amplified by nested PCR and it is feasible to use for mutation screening in the entire sequence of PKD1 cDNA, reducing the numbers of PCR reactions and SSCP gels required for the analysis. In the application of long RT-PCR and mutation screening by MRF-SSCP in our study, we could detect PKD1 mutations in 5 out of 10 families (50%). The limitation of detection rate may be due to low sensitivity of SSCP for mutation screening in long cDNA fragments (>300 bp) usually present after the digestion of PCR products with restriction enzymes and also probably due to the use of only single SSCP condition. However, this detection rate is comparable to that of protein-truncation test (PTT) which is about 52% [26, 27] although it is lower than that of DGGE [28–30] and DHPLC  which are about 60% and 70%, respectively. In the MRF-SSCP analysis, false-positive mobility shifts of the digested-cDNA fragments were found to be about 10%, which could be mainly eliminated by repeating the screening experiment or performing the analysis in more than one affected members in the same family. Although PTT method can screen for a relatively large cDNA fragments (~2 kb) and the detected alterations are almost all pathogenic mutations, its major disadvantage is that missense mutations, which are a significant source of PKD1 mutation  or small in-frame changes are not detected. Thus, it may not be an ideal screening method for detection of all types of PKD1 mutations. DGGE and DHPLC are the methods of choice provided that the equipments are available and the detection conditions are well optimized. Direct sequencing of the entire PKD1 gene is however very expensive if mutation screening is not initially performed and it may not be appropriate for such a large gene as PKD1.
PKD1 mutations reported so far in Thai families appear to be heterogeneous, similar to other ethnic origins worldwide. However, some degree of clustering of mutations in the regions encoding the PKD repeats and REJ domain of polycystin-1 is noticed (Figure 6) [19, 20, 31, 32]. The mutations that we identified in this and previous studies [19, 20] consisted of splicing, frameshift, in-frame and nonsense alterations. We have not yet observed missense mutations which have already been reported in Thai patients with ADPKD by Phakdeekitcharoen et al. [31, 32]. It is possible that our method has a low sensitivity for detecting missense mutation. However, as several single nucleotide polymorphisms (SNPs) could be identified (Table 4), we believe that our method is capable to also identify this type of mutation. It is likely that no detectable missense mutation in our study may occur by chance. No relationship between specific mutation and clinical manifestations was found from our data, similar to that observed in many other reports. With our long RT-PCR and nested PCR and the mutation screening by MRF-SSCP method or other modifications to increase screening efficiency such as the use of DHPLC , we expect to identify majority of PKD1 mutations, if not all, in our patients for the benefits of better understanding of pathogenesis of ADPKD, development of rapid molecular genetic diagnosis, and improvement of preventive measure for the complications related to the disease.
This study is the first report using the long RT-PCR method in combination with MRF-SSCP for mutation screening of PKD1 cDNA and total five mutations were identified within the reiterated region. Three mutations (splicing defect, frameshift alteration and in-frame deletion) are novel. Two mutations (splicing defect and nonsense change) are possibly de novo. PKD1 mutations reported so far in Thai patients with ADPKD are heterogeneous but mutations in a majority group of patients are still unknown, requiring further characterization.
We thank all ADPKD patients, family members, and laboratory personnel who participated and donated blood samples for the study. We also thank staff members of the Renal Unit, Department of Medicine, Faculty of Medicine Siriraj Hospital, for helping in the blood sample collection. This work was supported by the Thailand Research Fund (TRF) Post-Doctoral Research Grant (to WT), Mahidol University and National Research Council of Thailand (NRCT) (to CL), and the National Center for Genetic Engineering and Biotechnology (BIOTEC), Thailand (to PY).
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