Human Molecular Genetics

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Human Molecular Genetics, 2000, Vol. 9, No. 3 447-452
© 2000 Oxford University Press

Genetic evidence for a trans-heterozygous model for cystogenesis in autosomal dominant polycystic kidney disease

Michael Koptides, Richard Mean, Kyproula Demetriou¹, Alkis Pierides¹ and C. Constantinou Deltas⁺

The Cyprus Institute of Neurology and Genetics, Department of Molecular Genetics, PO Box 3462, 6 International Airport Avenue, Ayios Dhometios, 1683 Nicosia, Cyprus and ¹Department of Nephrology, Nicosia General Hospital, Nicosia, Cyprus

Received 21 October 1999; Revised and Accepted 3 December 1999.

	ABSTRACT

TOP ABSTRACT INTRODUCTION RESULTS AND DISCUSSION MATERIALS AND METHODS REFERENCES

 
Polycystic kidney disease (ADPKD) is a condition with an autosomal dominant mode of inheritance and adult onset. Two forms of the disease, ADPKD1 and ADPKD2, caused by mutations in PKD1 and PKD2, respectively, are very similar, except that ADPKD1 patients run a more severe course. At the cellular level, ADPKD1 was first shown to be recessive, since somatic second hits are perhaps necessary for cyst formation. The near identical phenotype had suggested that ADPKD1 and ADPKD2 might have a similar pathogenesis and that the two gene products, poly- cystins 1 and 2, are part of a common developmental pathway. Work in transgenic mice showed that somatic loss of Pkd2 expression is necessary for renal cyst formation, and recently we showed that somatic mutations inactivating the inherited healthy allele were present in 9 of 23 cysts from a human ADPKD2 kidney, supporting a two-hit loss-of-function model for ADPKD2 cystogenesis. Here, we provide the first direct genetic evidence that polycystins 1 and 2 do interact, perhaps as part of a larger complex. In cystic DNA from a kidney of an ADPKD1 patient, we showed somatic mutations not only in the PKD1 gene of certain cysts, but also in the PKD2 gene of others, generating a trans-heterozygous state with mutations in both genes. One mutation in PKD1 is of germinal nature and the mutation in the PKD2 gene is of somatic nature. The implications of such a situation are enormous, not only for ADPKD, but also for many other conditions with phenotypic heterogeneity and age-dependent penetrance.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION RESULTS AND DISCUSSION MATERIALS AND METHODS REFERENCES

 
Autosomal dominant polycystic kidney disease (ADPKD) is very frequent, affecting ~1 in 1000 individuals, and is mainly a disease of the elderly. It is characterized by age-dependent penetrance, and end-stage renal failure (ESRF) affects ~50% of patients by the age of 60 years. Phenotypic heterogeneity of inter- and intrafamilial nature is only partly explained by the genetic and allelic variability. Also, at the organism level, this variability is exemplified by the fact that only a very small percentage of renal epithelial cells (1–5%) develop focal cysts, knowing that all of them inherit the same germinal defect (1). In an effort to investigate the aetiology of focal cyst formation, data collected from several independent laboratories showed that a crucial step in cystogenesis is the somatic inactivation of the wild-type copy of the allele, which is inherited from the healthy parent, in renal or liver epithelial cells (2–7). Similar findings were obtained from experiments in murine mutant Pkd1 and Pkd2 models (8,9). These data suggested that both ADPKD1 and ADPKD2 are recessive at the cellular level, and explained part of the phenotypic variability and the age-dependent penetrance, since somatic mutagenesis is a stochastic event. However, it has been demonstrated that a large proportion of ADPKD1 and ADPKD2 cysts show strong immunoreactivity for polycystin 1 and 2 (10–13). Several factors could be responsible for these observations: (i) although a missense mutation may disrupt the function of the protein, the antibody used to detect polycystin 1 and 2 will react as the domain is still present; (ii) it is possible that a mutation inactivating polycystin 1 may increase its half-life; (iii) a trans-heterozygous state may also be responsible for cystogenesis. A trans-heterozygous state is the situation where a cell may be heterozygous for mutations on both genes, PKD1 and PKD2.

In the work presented here, we provide the first genetic evidence supporting the trans-heterozygous model as an alternative mechanism leading to focal cyst formation in ADPKD.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION RESULTS AND DISCUSSION MATERIALS AND METHODS REFERENCES

 
The similarity of ADPKD1 and ADPKD2 phenotypes prompted many investigators to hypothesize that the respective gene products might be components of a common developmental pathway, and that there could be interaction between them. Evidence for such interaction through their short intracytoplasmic domains with a coiled-coil structure was provided by two reports (14,15). This explained further why mutations in either PKD1 or PKD2 can cause almost identical symptomatologies.

Here, we investigated the trans-heterozygous hypothesis on a kidney from a patient of a Cypriot family, CY1609 (Fig. 1). The germinal mutation remains unknown, but DNA linkage analysis with marker 3'HVR, which is linked to the PKD1 gene, gave a positive LOD score of 1.85 at recombination fraction = 0.0. Also, linkage analysis with PKD2 marker D4S231 excluded linkage (the LOD score was –2.19 at = 0.0). Screening of the entire PKD2 gene by single-strand conformation polymorphism (SSCP) analysis did not reveal any PKD2 germinal mutation in this family.

View larger version (22K): [in this window] [in a new window]   Figure 1. Pedigree of family CY1609. The inheritance of haplotypes around the PKD1 and the PKD2 loci is shown. Statistical evaluation of the data showed that the disease phenotype is due to a PKD1 inherited DNA mutation which remains unknown (haplotype indicated by a left-pointing arrowhead). Two members, one in generation IV and one in generation V, have not been tested clinically, but based on the molecular analysis they are expected to develop the disease since they have inherited the affected haplotype. The kidney investigated was removed from the patient indicated by a right-pointing arrow.

 
We examined DNA from 23 cysts for loss of heterozygosity (LOH) and for mutations in both the PKD1 and PKD2 genes. Only one cyst showed LOH for PKD1, which extended from exon 25 within the gene and proximally up to marker SM6 (data not shown). Another four cysts gave different SSCP patterns in exons 24, 25, 31 and 39 that were of somatic acquired nature. We characterized fully the mutation in exon 24, which was a deletion of nine nucleotides, removing three amino acid residues. Those were phenylalanine, isoleucine and serine at positions 2979–2981 (8934–8942del), affecting the REJ module in the extracellular domain of polycystin 1 (Fig. 2A). Taking advantage of the polymorphisms G9195C and T9196C in exon 25 (M. Koptides and C. Constantinou Deltas, unpublished data), we proved that this somatic mutation occurred on the allele inherited from the healthy parent (data not shown). The occurrence of somatic mutations on the inherited normal allele of cystic DNA corroborates data described previously by several laboratories (2–7).

View larger version (16K): [in this window] [in a new window]   Figure 2. DNA sequences around the somatic mutations identified in the PKD genes of certain cysts. The upper sequences in (A) and (B) show the normal alleles and the lower sequences show the mutant alleles. The numbers above the first nucleotide of the sequence shown correspond to the cDNA position (nucleotide number 1 is the A residue of the first ATG codon). (A) Mutation in exon 24 of the PKD1 gene. The underlined bold sequence of nine nucleotides represents the deleted part. This mutation results in the deletion of three amino acids, indicated with asterisks. The lower case letters represent intronic sequence. (B) Mutation in exon 1 of the PKD2 gene of three separate cysts. The underlined bold sequence denotes the stretch of six Cs, wherein the C insertion occurred. This mutation is expected to cause a translation frameshift, leading to the incorporation of 22 novel amino acids before a new stop codon is encountered.

 
A striking finding was that three cysts showed an aberrant identical SSCP pattern in exon 1 of PKD2. Interestingly, when sequenced, all three cysts showed the exact same mutation, 197–203insC (data not shown), which we had previously described in seven different cysts originating from both kidneys of an ADPKD2 patient (6). The mutation represents a de novo insertion of a cytosine residue anywhere between nucleotides 197 and 203, which is predicted to introduce a translation frameshift (Fig. 2B). Based on those findings, we had suggested that this stretch of six Cs in exon 1 may represent a hot spot.

It was possible to suspect that this finding may have been an unfortunate DNA contamination event; however, it should be emphasized that extreme caution was exercised, both during cyst dissection and also during polymerase chain reaction (PCR) amplification of the various gene sequences. All PCR reactions were set up in a separate room within an ultraviolet-irradiated self-contained compartment, and blank samples containing everything but DNA were included in all series of amplifications. It should be noted that the three cysts that showed the above mutation were among many others, which were analysed simultaneously side by side, and that had shown no mutation in that same region. In addition, utilizing the intragenic polymorphism R28P (nucleotide 83 G/C) (6) we proved the different and independent origin of the cystic DNA of the case presented here. The patient we reported on previously was heterozygous G/C at position 83, whereas the present patient is homozygous G/G. Any perceptible con- tamination would have made evident the C allele, which was not the case (Fig. 3). Similarly, contamination with genomic DNA from the cysts that we analysed in our previous work was also excluded using polymorphic marker SM6 of proximal location, and KG8 in the 3'-untranslated region of PKD1 (data not shown). Interestingly, the mutation 197–203insC was recently reported independently by another group in a patient of British origin. In that case it was of a germinal nature and showed segregation through the respective family (16). This finding further suggests that the stretch of six Cs in exon 1 represents a hot spot.

View larger version (36K): [in this window] [in a new window]   Figure 3. Examination and exclusion of contamination during PCR amplification of PKD2 gene sequences. The photograph shows the Bsp1286I restriction enzyme digest of PCR products encompassing the polymorphic site at nucleotide position 83 of exon 1 (R28P) (6). The presence of C at position 83 creates a recognition site for Bsp1286I. Lane 1, peripheral blood leukocytic DNA from the index patient; lanes 2–4, genomic DNA from the three renal cysts of the index patient, which had undergone a somatic mutation in the PKD2 gene; lane 5, peripheral blood leukocytic DNA from the patient described previously, who was heterozygous G/C for the examined polymorphism (6); lane 6, control DNA with a G/C genotype; lane 7, control DNA with a C/C genotype; lane M, X174, HaeIII digest, molecular weight markers. Should there be any contamination during analysis of the cystic DNA of the index patient described here, one would expect to see molecular evidence of the C allele in lanes 2–4, which is not the case.

 
It did not escape our attention that, considering all available data, only a relatively small percentage of cystic cells have been shown to experience LOH or somatic mutations involving the inherited wild-type allele. The range of cysts showing this phenomenon was 17–24% in ADPKD1 and 22–43% in ADPKD2 cases. One explanation might be that not all cysts are the result of LOH or somatic mutations involving the other PKD allele. As suggested in this report, the second PKD1 allele may continue being normal and a trans mutation in one of the PKD2 alleles may be the triggering event, according to a model we developed (Fig. 4). There may also be cysts developed because of a germinal PKD1 or PKD2 mutation and somatic hits that affect other critical components of the polycystin complex (17). Apart from somatic inactivation of other genes, it was shown in experiments with animals that transgenic overexpession of c-myc in mice may trigger cyst formation (18,19), whereas several other modifier genes have been mapped to different locations in the mouse genome (20–23). Equally important are recent reports proposing that a polymorphic dinucleotide repeat in intron 1 of the epidermal growth factor receptor is a mediator of cyst formation in human polycystic kidney disease (24,25). Such scenarios would be compatible with the low rate of PKD1 or PKD2 somatic mutations reported and with the strong immunoreactivity found in most of the cystic epithelia examined from patients with ADPKD1.

View larger version (31K): [in this window] [in a new window]   Figure 4. Polycystin complex structures: schematic representation of a probable subset of structures of polycystin complex, as previously described (17). A total of 16 structures can be drawn, considering all possibilities of mutations in PKD1 and PKD2 genes. Structures I, II and III are possible combinations in the case of a germinal mutation in a cell that has not yet undergone a somatic mutation. It is assumed that the germinal mutation is in the PKD1 gene. A corresponding set of structures can be envisioned with a germinal mutation in PKD2 (data not shown, for reasons of simplicity and economy of space). All but structure I are predicted to be non-operational or partly operational, and perhaps unstable and amenable to degradation. A dominant-negative effect is evident, whereby one mutant molecule in a complex causes the inactivation of additional normal molecules that participate in the complex. Structures IV and V predict a PKD1 germinal and a PKD1 somatic mutation. Structure VIII predicts the presence of the wild-type polycystin 1 in a complex with two somatically mutated polycystin 2 molecules. The ‘P’ on polycystin 1 molecules predicts a putative phosphorylation site. On the background of a PKD1 germinal mutation in ADPKD1, a somatic defect in PKD2 of a certain cell, or vice versa, will give rise to trans-heterozygosity and lead to the formation of complexes such as structures VI, VII and VIII, and other combinations that can be envisioned. Most complexes will have one or more mutated polycystin 1 or 2 molecules. At the same time it is predicted that based on the model presented 1/16 will be normal (1/2 of structure I), a quantity, however, that most probably is not adequate to support normal renal cell and kidney development. It is evident that a dominant-negative effect or a ‘protein suicide’ effect takes place, with grave results.

 
To our knowledge, there is no precedent to the trans-heterozygous model as a mechanism for human disease development. Our findings raise the possibility that other complex phenotypes may be the result of trans-heterozygosity for mutations in related and perhaps cooperative recessive gene systems. In Drosophila melanogaster, a trans-heterozygous situation for two recessive mutations, the multiple wing hair and flare3, has been exploited for developing the wing spot test, which allows the identification of genotoxic substances (26).

The data presented provide more direct genetic evidence for cooperative interaction of polycystins 1 and 2, and support the hypothesis that somatic mutations in either PKD gene can trigger cyst formation on the background of an inherited PKD1 or PKD2 defect. Based on the experimental evidence generated to date, Murcia et al. (17) devised a model of the polycystin complex according to which two polycystin 2 molecules interact with each other and each one interacts separately with a polycystin 1 molecule (for details, see ref. 17). Mutated polycystin 1 or 2 may not be functional or may be partly functional and participate in the formation of a complex that is not or is partly operational. The model predicts that in the presence of only a germinal mutation 25% of the complexes will be normal and operational (Fig. 4, structure I). This may or may not be able to support normal development. A somatic mutation that inactivates the second healthy PKD1 or PKD2 allele in a certain renal cell abolishes the 25% normal complexes and converts them to doubly mutant (Fig. 4, structure III). In the case of trans-heterozygosity, many more combinations are possible, all of them predictably leading to compromised function (Fig. 4, structures IV–VIII).

Our model is rather speculative at this stage, but nevertheless the hypothesis presented here is worthy of further investigation as, if the data are further reproduced and confirmed, it may change the way of thinking as regards other late-onset diseases and aging processes that may be instituted or modified by putative somatic mutations in various cooperative gene systems.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION RESULTS AND DISCUSSION MATERIALS AND METHODS REFERENCES

 
Patient information
The patient belongs to a family, CY1609, that was previously investigated by DNA linkage analysis and had shown weak evidence for linkage to PKD1. No PKD2 markers had been examined (27). For the needs of the present study, blood samples from more family members were collected and the haplotype analysis was extended using additional PKD1 and PKD2 markers. Statistical evaluation was as described previously (28). The kidney was obtained during surgical resection as a result of medical advice relating to its huge size, which was due to hundreds of large cysts.

Isolation of cyst epithelial cells and genomic DNA extraction
This was an impressively large kidney with hundreds of cysts of variable size. Renal epithelial cells from the inner lining wall of each cyst and subsequent DNA extraction were performed as described with precautions to avoid contamination with non-cystic tissue (3,5).

Search for LOH and somatic mutations
LOH was tested with informative flanking and intragenic markers as described (5,6). Both PKD1 and PKD2 coding sequences and sequences at the exon–intron boundaries were screened by SSCP analysis, using primers and conditions as described previously (5,6,29). Especially for the screening of part of the PKD1 gene within the duplicated region, long-range PCR was attempted with the reverse unique primer anchored in exon 34 and a forward primer in exon 23 (5). All PCR reactions were set up in a separate room within an ultraviolet-irradiated self-contained compartment (Template Tamer; COY, MI). DNA sequencing was performed either directly on PCR products or after cloning into a plasmid vector. Multiple clones from two separate PCR products were sequenced for verification of the 9 bp deletion reported in the PKD1 gene of one cyst.

	ACKNOWLEDGEMENTS

 
We are grateful to Drs G. Kyriakides and M. Hadjigavriel of the Paraskevaidion Surgical and Transplantation Center for making available to us the kidney removed from the patient investigated. Also, we thank M.-D. Georgiou and P. Vorkas for technical assistance. This work was funded by Cyprus Telethon 98 and a grant by the Cyprus Kidney Association to C.C.D.

	FOOTNOTES

 
⁺ To whom correspondence should be addressed. Tel: +357 2 392655; Fax: +357 2 358237; email: deltasco@mdrtc.cing.ac.cy

	REFERENCES

TOP ABSTRACT INTRODUCTION RESULTS AND DISCUSSION MATERIALS AND METHODS REFERENCES

 
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