Mutations in the PKD2 gene on the long arm of chromosome 4 are responsible for ~15% of cases of polycystic kidney disease. Perhaps the only difference from the more common ADPKD1 cases is the rate of progression of cystic changes, and the age of onset, which is 10-15 years later for the ADPKD2 form. In Cyprus there are at least three large families, documented by molecular linkage analysis, that map to the PKD2 locus. For two of them the defects were recently shown to be nonsense mutations at positions arginine 742 and glutamine 405. In this report, we describe the mutation in the third family, CY1602. For this, the entire coding sequence was systematically screened by single strand conformation analysis and heteroduplex formation. A novel mutation was identified in exon 2 where a new cytosine residue was inserted immediately after codon 231 (231insC). It causes a translation frameshift and is expected to lead to the introduction of 37 novel amino acids before the translation reaches a new STOP codon. It is the most amino terminal mutation reported to date, and based on the protein's modeled structure, is predicted to be within the first transmembrane domain. It is the fourth PKD2 mutation reported thus far, and the first which is not a nonsense mutation.
The most common form of Autosomal Dominant Polycystic Kidney disease (ADPKD) is caused by mutations in the PKD1 gene on chromosome 16 at 16p13.3, and it accounts for ~85% of cases. The second gene, PKD2, maps to chromosome 4q21-23, and accounts for ~15% of affected families (1 -3 ). A third much rarer gene, PKD3, has been implicated in some families and is of unknown chromosomal location (4 -7 ). The principal manifestation for all forms of polycystic kidney disease (PKD) is the bilateral formation of fluid filled-cysts in the kidneys and, in a smaller percentage of cases, in the liver and other organs. Hypertension is also a common feature in families with PKD.
The PKD1 gene has been recently cloned and mutations have been reported in affected families (8 -15 ). More recently, the PKD2 gene was identified and mutations were reported in three PKD2 families (16 ). Most of the mutations described in the PKD1 gene and all three mutations described for the PKD2 gene are nonsense mutations, leading to the predicted production of truncated proteins. In PKD1, small or large deletions and splice variants, one duplication mutation and a few missense mutations have also been identified. Here we report on a four-generation Cypriot family where the disease causing mutation is a single nucleotide insertion in the second exon of the PKD2 gene.
A series of primer pairs were designed encompassing all exons of the PKD2 gene and were used for SSCP screening for mutations of candidate patients (T. Hayashi, T., Mochizuki, D.M. Reynolds, W. Guanqing, C. Yiqiang and S. Somlo, in preparation). This approach was used in our laboratory initially on four samples. Two of them were from PKD2 patients in family CY1602, one was from a healthy individual in the same family, and the fourth sample was the 175 kb P1 genomic clone, p157n2, that contains nearly all of the coding PKD2 gene (16 ). PCR products for several exons were electrophoresed simultaneously on non-denaturing polyacrylamide gels for SSCP analysis as described in Materials and Methods. One DNA variant was identified in the IF5/IR20 PCR product encompassing exon 2, and it co-segregated with the disease phenotype in family CY1602 (Fig. 1 ). This variant can also be detected by heteroduplex analysis on MDE gels (Fig. 2 ).
PKD is a heterogeneous inherited disease with at least three genes responsible for the development of similar pathology. Two of these, PKD1 and PKD2, on chromosomes 16 and 4 respectively, were cloned and sequenced in the recent past. The PKD1 gene, mutations in which cause the most common form of the disease, is encoded in 46 exons and its inherent complexity makes routine mutational analysis technically prohibitive. The main reason is that ~80% of its sequence at the 5'-end is duplicated elsewhere on chromosome 16, thereby rendering specific PCR amplification of that region almost impossible (8 ,17 -19 ).
In contrast to the above situation, screening of the entire PKD2 coding sequence is much easier, faster and straightforward. The streamlined analysis by SSCP, employing three different electrophoretic conditions, significantly reduces the probability of missing DNA variations (13 ,20 ). This approach may indeed be more cost effective and efficient by using one or two individuals of a given family, than trying to collect all necessary samples for performing classical, complete family linkage analysis, when presymptomatic diagnosis is sought by concerned members. Knowing the exact molecular defect in this family allowed more definitive presymptomatic diagnosis to be made in young individuals where clinical examination would be equivocal. Thus, individual IV-6 is expected to develop the disease, therefore he will be more carefully monitored for early complications. Equally important is the diagnosis of other members of the fourth generation who did not inherit the disease gene, and they and their parents are relieved of the anxiety accompanying their status uncertainty.
The mutation identified in family CY1602 predicts the production of a truncated protein of 268 amino acids long, whereas the full length protein is 968 amino acids long (16 ). Thirty-seven amino acids at the end are not normally encoded by the gene, but they are expected to be introduced as a result of the nature of the mutation that causes a translation frameshift. The mutation is topographically predicted to be located at the beginning of the first transmembrane domain, and possibly preventing it from reaching its correct final destination. This should prevent it from exerting its normal function which, based on its structure, is hypothesized to be that of an ion channel or pore (16 ). Although it has been possible by allele specific oligonucleotide hybridization to demonstrate the presence of mutant mRNA in peripheral blood leucocytes of patients, it is not known how stable this mRNA is, or whether it is successfully translated to a stable product. Also, it is difficult to speculate about the disease causing mechanism.
The family described here was previously haplotyped with polymorphic markers around both the PKD1 and PKD2 genes and it was shown to be linked to the PKD2 locus (21 ) (Family CY1602). The proband reached ESRD and commenced haemodialysis at the age of 70. At age 54 he had a mild cerebrovascular accident and he presented at 64 with hypertension. Patient III-7 of the next generation, presented at age 49 with hypertension, fatigue, chronic renal failure, large kidneys and serum creatinine of 1.76 mg/dl. Three years later her creatinine was raised to 8.3 mg/dl and she started haemodialysis at 52. She was successfully transplanted 6 years later. She also had liver cysts. Her mother, II-5, died at age 64 from ESRD, after a short period of uraemia, at a time when haemodialysis was not available in the country. Patient III-1 presented at age 46 with hypertension, renal cysts and normal kidney function. Patient IV-6, at age 23 is well with no hypertension but multiple cysts in the left kidney and no liver cysts.
The PKD2 gene is encoded in 15 exons. Fourteen pairs of polymerase chain reaction (PCR) DNA amplification primers that are located in the flanking intronic sides of exons 2-15, and three pairs covering the first exon, were used for mutation screening by SSCP and heteroduplex analysis in several patients. The complete set and sequence of primers is described elsewhere (T. Hayashi, T. Mochizuki, D.M. Reynolds, W. Guanqing, C. Yiqiang and S. Somlo, in preparation. Before publication the primers are available by S. Somlo upon request). The SSCP procedure was essentially as described by Sheffield et al. (20 ) and modified by Neophytou et al. (13 ). Briefly, the PCR products were examined under three separate electrophoretic conditions for maximizing the probability of detecting DNA variations. The PCR products were brought to 40% formamide, denatured at 95oC and loaded onto a 6% non-denaturing polyacrylamide gel. The three different electrophoretic conditions for SSCP were: gels with 49:1 acrylamide:bis-acrylamide, were electrophoresed at 50-100 W in a 4oC cold room, for 2.5 h; gels composed of 29:1 acrylamide:bis-acrylamide were electrophoresed either at 50-100 W for 2.5 h, in a 4oC cold room, or at a very low power (1-4 W) at room temperature for 17 h. All gels were made and run in 0.5* TBE buffer (1* TBE buffer = 0.09 M TRIS-borate, 0.002 M EDTA, pH 8.3). After electrophoresis, the gels were fixed in 10% ethanol/10% glacial acetic acid solution, dried and exposed to Kodak X-ray film. The primers with which the mutation was identified in this family were IF5: 5'-AAATGATATCTTTTCTTTTCTTCA-3', and IR20: 5'-AACTTTCCCATTAGTGCAAG-3'. Annealing was at 55oC. The PCR product was 188 bp encompassing exon 2 of 114 bp. Heteroduplex analysis for confirmation was performed using Hydrolink Mutation Detection Enhancement gels (MDE), following the manufacturer's instructions (AT Biochem, Malvern, PA, USA).
The PCR products that showed a variation by SSCP analysis were directly sequenced. Sequencing was performed on asymmetrically amplified single strand DNA with the T7 DNA polymerase kit from Pharmacia. Total RNA extraction was performed on peripheral blood leucocytes from fresh blood using the RNAzol kit from Wak-Chemie Medical (Bad, Homburg, Germany). Reverse transcription (RT) for first strand cDNA synthesis was performed using an oligo-dT primer and a kit from Pharmacia. DNA amplification by PCR was performed with Taq DNA polymerase and other reagents from Pharmacia and Perkin-Elmer Cetus, on an MJ Research thermocycler.
Identification of the mutation on cDNA of normal and affected individuals of family CY1602, was performed by hybridization with allele specific oligonucleotides. For this, two 19mers oligonucleotides were prepared encompassing the site of the mutation. The normal primer was ASO-231insC.W: 5'-CCTTTTTCTCATAGTCTTG-3' and the mutant primer was ASO-231insC.M: 5'-CTTTTTCTCCATAGTCTTG-3'. PCR products of the relevant region were first size-fractionated by electrophoresis on 2% agarose gels in duplicate and transferred by capillary action on to Hybond-N+ membranes (Amersham). The oligos were end-labelled with the use of T4 polynucleotide kinase and [32P-[gamma]]ATP. They were used for hybridization without prior purification. Hybridization was overnight at 45oC in 5* SSPE/5* Denhardt's solution/0.5% SDS. It was washed once with 2* SSPE/0.1% SDS for 10 min at room temperature, and once with 2* SSPE/0.1% SDS at 45oC for 10 min. The final wash was with 1* SSPE/0.1% SDS for 10 min at 45oC (1* SSPE = 0.18 M NaCl, 0.01 M sodium phosphate, 0.001 M EDTA, pH 7.7). Membranes were exposed to Kodak X-ray film as needed.
This work was partly funded by the Cyprus Ministry of Health, the Cyprus Kidney Association and grants from NIH (DK48383, SS and CCD) and the March of Dimes Birth Defects Foundation (SS).
ADPKD, autosomal dominant polycystic kidney disease; ESRD, end-stage renal disease; PKD1/2, polycystic kidney disease 1/2 gene; RT-PCR, reverse transcription/polymerase chain reaction; SSCP, single strand conformation polymorphism; ASO, allele specific oligonucleotide; bp, base pairs.
*To whom correspondence should be addressed. Tel: +357 2 392655; Fax: +357 2 358237; Email: deltasco@mdrtc.cing.ac.cy
Human Molecular Genetics
Pages
Introduction
Results
Discussion
Materials And Methods
Clinical information
Single strand conformation polymorphism analysis (SSCP) and heteroduplex analysis
DNA sequencing, RNA extraction and RT-PCR
Allele specific oligonucleotide hybridization
Acknowledgements
Abbreviations
References
REFERENCES
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