A novel frameshift mutation (2436insT) produces an immediate stop codon in the autosomal dominant polycystic kidney disease 2 (PKD2) gene

Diana M. Iglesias1,4, Dolores Telleria2, Miguel Viribay2, Mariana Herrera3, Viviana A. Bernath3, Alberto R. Kornblihtt1, Rodolfo S. Martin4 and José Luis San Millán2

1 Laboratorio de Fisiología y Biología Molecular, Departamento de Ciencias Biológicas, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Argentina, 2 Unidad de Genética Molecular, Hospital Ramón y Cajal, Madrid, Spain, 3 Biología Molecular Diagnóstica SA, Buenos Aires, Argentina and 4 Instituto de Investigaciones Médicas A Lanari, Universidad de Buenos Aires, Argentina

Correspondence and offprint requests to: Rodolfo S. Martin, Instituto de Investigaciones Médicas, Alfredo Lanari, Medical School, University of Buenos Aires, Combatientes de Malvinas 3150, 1427-Buenos Aires, Argentina.



   Abstract
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Background. Autosomal dominant polycystic kidney disease (ADPKD) is a genetically heterogeneous disorder that can be caused by mutations in at least three different genes. Several mutations have been identified in PKD1 and PKD2 genes. Most of the mutations found in PKD2 gene are predicted to cause premature termination of the protein.

Methods. We analysed an Argentinian family characterized previously as PKD2. The PKD2 gene was amplified from genomic DNA using 17 primer pairs and the products were analysed by heteroduplex analysis. PCR products that showed a variation by heteroduplex analysis were sequenced directly. The mutation was confirmed by sequencing relatives. The segregation of the mutation in this family was verified by restriction endonuclease digestion of PCR products obtained from genomic DNA of all family members.

Results and conclusions. Here, we report a novel mutation present in an Argentinian family characterized as PKD2 by linkage analysis. The mutation, shared by all affected members of the family, is a thymidine insertion at position 2436 of the gene, which results in a translation frameshift and creates an immediate stop codon. This mutation is expected to lead to a truncated protein that lacks the interacting domain with the PKD1 gene product. The thymidine insertion abolished a Ddel restriction site, allowing a rapid test for detection of PKD2 carriers in the family.

Keywords: heteroduplex analysis; mutational analysis; polycystic kidney disease type 2



   Introduction
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Autosomal dominant polycystic kidney disease (ADPKD) is an heterogeneous genetic disorder with at least three different genes responsible for the development of the disease. The PKD1 form accounts for the majority of ADPKD cases and the PKD1 gene has been identified on chromosome 16p13.3 [1]. The PKD2 gene has been located to chromosome 4q21–23 and accounts for ~15% of cases [2]. There is a third group of PKD patients with no linkage to either chromosome 16 or chromosome 4 [3] and with no chromosomal location described to date. Clinically, the disease is characterized by the formation of bilateral fluid-filled cysts in the kidneys and other organs such as the liver, spleen and pancreas, typically leading to end-stage renal disease (ESRD). Intracranial aneurysms and hypertension are also major manifestations of the disease [4].

In Argentina, ADPKD was the underlying cause of renal failure in 5.7% of 2608 patients that began chronic dialysis in the period 1993–1995 [5]. These figures are lower than those reported in other populations, in which the disease accounts for 8–10% of ESRD [4]. Fifty-one per cent of ADPKD patients had entered into ESRD by the age of 60 and 70 years, in an analysis of 57 patients from Buenos Aires [6]. No attempt was made to differentiate PKD1 from PKD2 in these studies. In this regard, it is known that PKD1 patients show an increased risk of progression to renal failure, the mean age of death or onset of ESRD being 53 years for PKD1 and 69.1 years for PKD2 [7].

Sequencing of two ADPKD genes, PKD1 and PKD2, has recently been completed [810] allowing mutation screening in affected individuals [1113]. Several mutations have been described for PKD2, with no evidence of clustering. They include 11 small intragenic deletions [1416], five splicing defects [15], 12 nonsense mutations [10,1416], five insertions of one nucleotide [1518] and two missense mutations [15,16].

Here, we report an insertion of a nucleotide at position 2436 within a large PKD2 family characterized previously [19], which is expected to lead to a truncated protein. This novel mutation was present in all the affected members of the family and is the first described in the Argentinian population.



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Clinical details
The family described here has been genotyped previously using polymorphic markers close to both the PKD1 and PKD2 genes and shown to be linked to the PKD2 locus [19] (family 16012). In this family, ADPKD can be traced through four generations, with 12 individuals known to be affected. All members carrying the disease-associated haplotype had renal cysts, independent of age. Two individuals in generations I and II (I, 1 and II, 2, Figure 1Go) reached ESRD at 80 and 68 years of age, respectively. Individuals II, 3 and II, 5 (Figure 1Go) showed serum creatinine values of 2.4 and 1.3 mg/dl at 65 and 66 years of age, respectively. Serum creatinine values in the rest of the affected individuals (shown in Figure 1Go) were less than 1 mg/dl, the eldest member (III, 3) being 33 years old. These data resemble those reported previously [7], with age at ESRD higher in PKD2 than in PKD1. Hepatic cysts and mild arterial hypertension were also detected in this family, at a frequency depending on age at the time of diagnosis. No history of nephrolithiasis episodes was found. Lastly, an atrial myxoma was diagnosed and successfully operated in an additional case (not shown in Figure 1Go) [20].



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Fig. 1. Segregation of the mutated allele with the disease phenotype in family 16012. The wild-type allele is cleaved by Ddel at C^TGAG, giving two fragments of 122 and 113 bp (bottom). The mutation (CTTGAG) abolishes the restriction site, giving an unique fragment of 235 bp (164 bp corresponding to exon 13 and 44+27 bp corresponding to part of the adjacent introns). Filled symbols are affected individuals, open symbols are unaffected individuals.

 
Mutation screening, DNA sequencing and restriction analysis
The PKD2 gene comprises 15 exons. The entire coding region was amplified from genomic DNA using 17 primer pairs as described previously [21]. For heteroduplex analysis, samples were denatured by heating at 95°C for 5 min and then allowed to cool at 37°C for at least 1 h. The reaction products were electrophoresed through 0.5xMutation Detection Enhancement gels (MDE, FMC), with 15% (w/v) urea, in TBE buffer at 250 V for 16–24 h. Gels were stained with ethidium bromide and photographed under UV light. PCR products showing a variation by heteroduplex analysis were sequenced directly in both directions using the PKD2 exon 13 primers IF3 and IR4 as sequencing primers [21]. The mutation was confirmed by sequencing three other relatives (two affected and one non-affected) and several recombinant plasmids obtained by cloning the exon 13 PCR product of an affected patient in a T-vector plasmid (pMos Blue, Amersham). Segregation of the mutation in this family was verified by restriction endonuclease digestion of PCR products obtained from genomic DNA of all family members. After amplification of exon 13 with primers IF3 and IR4, the PCR product was digested with Ddel, and DNA fragments were analysed by electrophoresis in 3% NuSieve (FMC) agarose gels.



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 Abstract
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 Subjects and methods
 Results
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 References
 
Previously, we characterized a four-generation Argentinian family (16012) as PKD2 by linkage analysis in a population study of ADPKD in Argentina [19]. In order to detect and characterize the mutation responsible for the disease in this family, genomic DNA from a patient was amplified by PCR with series of 17 primer pairs encompassing all exons of the PKD2 gene. The PCR products were screened for mutations by heteroduplex analysis on MDE gels, as described in Materials and methods. Heteroduplex formation was only identified in the PCR product corresponding to exon 13 (Figure 2Go), in which it appeared only in the affected members assayed (Figure 2Go). Direct sequencing of the PCR fragments from one normal and one affected individual was performed using PCR primers for exon 13. The sequences were identical in both samples until nucleotide 2436 of the coding DNA (considering the first A of the proposed methionine translation start codon as nucleotide 1), at which point a complex profile of double peaks appeared in the patient sample, suggesting that a frameshift had occurred in one allele. Closer inspection of the sequence (Figure 2Go) shows that the frameshift was the result of a single thymidine insertion at that position. The mutation was confirmed by sequencing both strands from two other affected individuals and by cloning the mutant allele into a bacterial T-vector plasmid. This mutation, 2436insT, leads to the creation of an immediate stop codon due to a reading frameshift (Figure 2Go). Interestingly, this 2436insT mutation abolishes a Ddel restriction site, and this enzyme was used as a rapid test to confirm the mutation in other members of this family. Genomic DNA was amplified with primers IF3 and IR4 [21], giving a PCR product of 235 bp. After digestion with Ddel, all normal individuals gave two fragments of 122 and 113 bp, while mutation carriers gave an additional fragment of 236 bp (Figure 1Go), confirming that the 2436insT mutation was responsible for the development of the disease in this family.



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Fig. 2. Characterization of mutation 2436insT in exon 13 of family 16012. (I) Heteroduplex analysis of DNA samples from two affected members (A), one non-affected (N) and one control individual (C). (II) DNA sequence analysis (from direct sequencing of PCR products) of the PKD2 gene exon 13 from an affected individual. The novel stop codon (TGA in codon 813) is underlined. The arrow indicates the location of the thymidine insertion.

 


   Discussion
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
ADPKD is genetically heterogeneous and involves at least three different genes: PKD1 on chromosome 16, PKD2 on chromosome 4 and a third, or more, genes of unknown location. Genetic heterogeneity is responsible for phenotypic variability, but different expression of the disease within families has also been described [22]. The PKD2 gene has been located to chromosome 4q21–23 and accounts for 15% of the ADPKD cases. This gene has been sequenced and characterized in the recent past [10]. Contrary to what happens with PKD1 gene, the PKD2 gene does not show duplicated regions elsewhere in the genome. Therefore, mutation studies of the whole gene are easy to accomplish by simple methods, such as heteroduplex analysis. The putative translation product of PKD2 is a 968-residue protein. The predicted model of polycystin 2, the product of the PKD2 gene, shows an integral membrane protein with six membrane-spanning domains, intracellular N- and C-terminal domains, and a putative calcium-binding EF domain in the cytoplasmic C-terminal section [10]. It has been shown that this C-terminal region of polycystin 2 is involved in homodimerization with other polycystin 2 molecules, as well as heterodimerization with polycystin 1 or even other proteins [23]. An important question is whether PKD2 mutations inactivate or generate a protein with a dominant negative or gain-of-function effect. The mutation reported here was located in exon 13 and is predicted to result in a frameshift, with premature translation termination of the PKD2 product immediately after codon 812. The truncated polycystin 2 is predicted to lack the cytoplasmic domain. Most of the mutations identified to date are scattered along the gene and are expected to produce truncated proteins, resulting in the loss of a large part of the C-terminal end. The nature and distribution of mutations, plus the lack of a clear phenotype/genotype correlation, indicate that they may inactivate the molecule. Recent data [24], showing evidence of somatic mutations in PKD2 cysts, support the hypothesis that PKD2 disease is likely to be caused by a two-hit mechanism, as described for PKD1 disease [25]. Because both polycystin 1 and 2 interact, it has been suggested that they may function via a common pathway. If so, a different rate of somatic second-hits in each gene could explain why the PKD2 phenotype is less severe than PKD1.

In summary, a novel mutation in an Argentinian family is described. It is characterized by a thymidine insertion at position 2436 of the gene, with translation frameshift and creation of an immediate stop codon.



   Acknowledgments
 
This work was supported by a grant from the Spanish Fondo de Investigaciones Sanitarias (JLSM) and partially supported by a grant from the Secretaría de Ciencia y Técnica Argentina (PID: PMT-SID 0262). DMI has a research fellowship from the University of Buenos Aires. We thank Dr J. E. Mamberti for his assistance in the clinical follow-up of our patients.



   References
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 Introduction
 Subjects and methods
 Results
 Discussion
 References
 

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Received for publication: 19. 2.99
Accepted in revised form: 25.11.99