Clinical utility of PKD2 mutation testing in a polycystic kidney disease cohort attending a specialist nephrology out-patient clinic

ABSTRACT: BACKGROUND: ADPKD affects approximately 1:1000 of the worldwide population. It is caused by mutations in two genes, PKD1 and PKD2. Although allelic variation has some influence on disease severity, genic effects are strong, with PKD2 mutations predicting later onset of ESRF by up to 20 years. We therefore screened a cohort of ADPKD patients attending a nephrology out-patient clinic for PKD2 mutations, to identify factors that can be used to offer targeted gene testing and to provide patients with improved prognostic information. METHODS: 142 consecutive individuals presenting to a hospital nephrology out-patient service with a diagnosis of ADPKD and CKD stage 4 or less were screened for mutations in PKD2, following clinical evaluation and provision of a detailed family history (FH). RESULTS: PKD2 mutations were identified in one fifth of cases. 12% of non-PKD2 patients progressed to ESRF during this study whilst none with a PKD2 mutation did (16-88 months of follow-up, p < 0.03). However, the overall age to development of CKD3 was not predicted by mutation status. A significant difference was found in age at ESRF of affected family members (non-PKD2 vs. PKD2, 54 yrs vs. 65 yrs; p < 0.0001). No PKD2 mutations were identified in patients with a FH of ESRF occurring before age 50 yrs, whereas a PKD2 mutation was predicted by a positive FH without ESRF. CONCLUSIONS: PKD2 testing has a clinically significant detection rate in the pre-ESRF population. It did not accurately distinguish those individuals with milder renal disease defined by stage of CKD but did identify a group less likely to progress to ESRF. When used with detailed FH, it also offers useful prognostic information for individuals and their families. It can usefully be offered to all but those whose relatives have developed ESRF before the sixth decade.     

Structural model of the TRPP2/PKD1 C-terminal coiled-coil complex produced by a combined computational and experimental approach

Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations in TRPP2 and PKD1, which form an ion channel/receptor complex containing three TRPP2 and one PKD1. A TRPP2 C-terminal coiled-coil trimer, critical for the assembly of this complex, associates with a single PKD1 C-terminal coiled-coil. Many ADPKD pathogenic mutations result in the abolishment of the TRPP2/PKD1 coiled-coil complex. To gain molecular and functional insights into this heterotetrameric complex, we computationally constructed a structural model by using a two-step docking strategy, based on a known crystal structure of the TRPP2 coiled-coil trimer. The model shows that this tetrameric complex has a novel di-trimer configuration: An upstream trimer made of three TRPP2 helices and a downstream trimer made of two TRPP2 helices and one PKD1 helix. Mutagenesis and biochemical analysis identified critical TRPP2/PKD1 interface contacts essential for the heteromeric coiled-coil complex. Mutation of these interface positions in the full-length proteins showed that these interactions were critical for the assembly of the full-length complex in cells. Our results provide a means to specifically weaken the TRPP2 and PKD1 association, thus facilitating future in vitro and in vivo studies on the functional importance of this association.     

Somatotroph pituitary adenoma with acromegaly and autosomal dominant polycystic kidney disease: SSTR5 polymorphism and PKD1 mutation

A 39-year-old woman with autosomal dominant polycystic kidney disease (ADPKD) presented with acromegaly and a pituitary macroadenoma. There was a family history of this renal disorder. She had undergone surgery for pituitary adenoma 6?years prior. Physical examination disclosed bitemporal hemianopsia and elevation of both basal growth hormone (GH) 106?ng/mL (normal 0?C5) and insulin-like growth factor (IGF-1) 811?ng/mL (normal 48?C255) blood levels. A magnetic resonance imaging scan disclosed a 3.0?cm sellar and suprasellar mass with both optic chiasm compression and left cavernous sinus invasion. Pathologic, cytogenetic, molecular and in silico analysis was undertaken. Histologic, immunohistochemical and ultrastructural studies of the lesion disclosed a sparsely granulated somatotroph adenoma. Standard chromosome analysis on the blood sample showed no abnormality. Sequence analysis of the coding regions of PKD1 and PKD2 employing DNA from both peripheral leukocytes and the tumor revealed the most common PKD1 mutation, 5014_5015delAG. Analysis of the entire SSTR5 gene disclosed the variant c.142C>A (p.L48M, rs4988483) in the heterozygous state in both blood and tumor, while no pathogenic mutations were noted in the MEN1, AIP, p27Kip1 and SSTR2 genes. To our knowledge, this is the fourth reported case of a GH-producing pituitary adenoma associated with ADPKD, but the first subjected to extensive morphological, ultrastructural, cytogenetic and molecular studies. The physical proximity of the PKD1 and SSTR5 genes on chromosome 16 suggests a causal relationship between ADPKD and somatotroph adenoma.     

Heterozygosity loss and somatic mutations in type I and II dominant autosomal renal polycystic kidney disease: evidence of a recessive mechanism at a cell level in cystogenesis

Autosomal dominant polycystic kidney disease (ADPKD) is a systemic disorder mainly characterized by renal cyst formation. Cysts in ADPKD are focal in nature, since only a small fraction of nephrons become cystic. The hypothesis that a second hit may be required for cyst formation has been proposed. This hypothesis suggests that inactivation of the inherited wild-type allele by a somatic mutation triggers cyst formation. In some cases, this second hit eliminates the normal allele and the affected cells remain with a single allele, which is the inherited mutated copy, and we only visualize one allele after the amplification by polymerase chain reaction; this is called loss of heterozygosity (LOH). In this study we have analysed the DNA isolated from epitehlial cells from 164 cysts of 8 kidneys affected by ADPKD type I and 30 cysts form a kidney affected by ADPKD type II. We have demonstrated the presence of LOH in 20.1% of PKD1 cysts and in 10% of PKD2 cysts. We have also found eight other different mutations in PKD2 cysts without LOH; so the percentage of somatic mutations in the PKD2 kidney reaches 36.6% of cysts. In conclusion, our data suggest that a recessive mechanism at the cellular level is implicated in cyst formation in the PKD1 and the PKD2 disease. The loss of both copies of the gene triggers the proliferation of a single cell, resulting in the cyst formation.     

Structural and molecular basis of the assembly of the TRPP2/PKD1 complex

Mutations in PKD1 and TRPP2 account for nearly all cases of autosomal dominant polycystic kidney disease (ADPKD). These 2 proteins form a receptor/ion channel complex on the cell surface. Using a combination of biochemistry, crystallography, and a single-molecule method to determine the subunit composition of proteins in the plasma membrane of live cells, we find that this complex contains 3 TRPP2 and 1 PKD1. A newly identified coiled-coil domain in the C terminus of TRPP2 is critical for the formation of this complex. This coiled-coil domain forms a homotrimer, in both solution and crystal structure, and binds to a single coiled-coil domain in the C terminus of PKD1. Mutations that disrupt the TRPP2 coiled-coil domain trimer abolish the assembly of both the full-length TRPP2 trimer and the TRPP2/PKD1 complex and diminish the surface expression of both proteins. These results have significant implications for the assembly, regulation, and function of the TRPP2/PKD1 complex and the pathogenic mechanism of some ADPKD-producing mutations.     

Polycystin 1 is required for the structural integrity of blood vessels

Autosomal dominant polycystic kidney disease (ADPKD), often caused by mutations in the PKD1 gene, is associated with life-threatening vascular abnormalities that are commonly attributed to the frequent occurrence of hypertension. A previously reported targeted mutation of the mouse homologue of PKD1 was not associated with vascular fragility, leading to the suggestion that the vascular lesion may be of a secondary nature. Here we demonstrate a primary role of PKD1 mutations in vascular fragility. Mouse embryos homozygous for the mutant allele (Pkd1(L)) exhibit s.c. edema, vascular leaks, and rupture of blood vessels, culminating in embryonic lethality at embryonic day 15.5. Kidney and pancreatic ductal cysts are present. The Pkd1-encoded protein, mouse polycystin 1, was detected in normal endothelium and the surrounding vascular smooth muscle cells. These data reveal a requisite role for polycystin 1 in maintaining the structural integrity of the vasculature as well as epithelium and suggest that the nature of the PKD1 mutation contributes to the phenotypic variance in ADPKD.     

Autosomal-dominante polyzystische Nierenerkrankung

Autosomal dominant polycystic kidney disease (ADPKD) is the most common genetic renal disease, and it is even one of the most common hereditary diseases in man. In 85% of the cases the disease is caused by mutations in the PKD1 gene, which encodes for the ciliary protein polycystin-1. A milder variant of the disease is caused by mutations in the PKD2 gene, which encodes for the calcium channel-related protein polycystin-2. The disease is characterized by the progressive development of innumerable cysts in both kidneys, which gradually replace the normal kidney tissue. In ADPKD patients the inexorable cyst growth leads to a progressive deterioration of renal function over decades, which ultimately can only be treated by renal replacement therapy or renal transplantation. Until now a causal treatment was not available, and treatment options were limited to regular clinical controls and treatment of complications (hypertension, cyst infections). Several promising clinical studies are currently examining new or even existing drugs as therapeutic options to retard cyst growth in ADPKD (vasopressin receptor-2 antagonists [V2RA], mammalian target of rapamycin [mTOR] inhibitors, somatostatin). It can be anticipated that ADPKD patients will benefit from these new treatment options in the near future.     

Historia natural de la poliquistosis renal autosómica dominante en Córdoba: utilidad de una base de datos para agrupar familias y mutaciones

Autosomal dominant polycystic kidney disease (ADPKD) is one of the main causes of end-stage renal disease. Today, the knowledge of its genetic base has made it possible to develop strategies that prevent the transmission of the disease.ObjectivesThe objective of the study was to analyze the natural history of ADPKD in the Province of Córdoba and to design a database that allows families and different mutations to be grouped.Patients and methodsAll patients (n = 678) diagnosed with ADPKD followed up in the Cordoba nephrology service are included. Various clinical variables (age and sex), genetic variables (mutation in PKD1, PKD2) and the need for renal replacement therapy (RRT) were retrospectively analyzed.ResultsThe prevalence was 61 cases per 100,000 inhabitants. Median renal survival was significantly worse in PKD1 (57.5 years) than in PKD2 (70 years) (Log-rank p = 0.000). We have genetically identified 43.8% of the population, detecting mutations in PKD1 in 61.2% and in PKD2 in 37.4% of cases. The most frequent mutation was detected in PKD2 (c.2159del) in 68 patients belonging to 10 different families. The one with the worst renal prognosis was detected in PKD1 (c.9893G>A). These patients required RRT at a median age of 38.7 years.ConclusionsThe renal survival of ADPKD in the Province of Córdoba is similar to that described in the literature. Mutations in PKD2 were detected in 37.4%. Our strategy allows us to know the genetic bases of our population with a great saving of resources. This is essential to be able to offer primary prevention of ADPKD through preimplantation genetic diagnosis.     

Interpretation of renal volume in autosomal dominant polycystic kidney disease and relevant clinical implications

Autosomal dominant polycystic kidney disease (ADPKD) is the most common life-threatening hereditary disease of the kidney. It presents with progressive enlargement of the kidneys with numerous cysts that distort the parenchyma and result in progressive decline in kidney function. Autosomal dominant polycystic kidney disease is genetically modified with the responsible genes localized to separate loci on chromosome 16 (PKD1 gene), accounting for the majority of ADPKD cases, and chromosome 4 (PKD2 gene), accounting for the remainder. This review discusses the current understanding of the pathogenesis of ADPKD, focusing on renal volume and its pivotal role on the manifestations of the disease. Specifically, activation of the renin-angiotensin-aldosterone system, hypertension, left ventricular hypertrophy, kidney function deterioration, pain, and hematuria are examined as consequences of renal volume increase. Recent developments on diagnostic modalities and criteria of the ADPKD are also discussed.     

Chronic pancreatitis with polycystic kidney disease: A rare coincidence?

IntroductionIn children, chronic pancreatitis (CP) is usually associated with anatomical anomalies of the pancreas and biliary tract or is genetically determined. Autosomal dominant polycystic kidney disease (ADPKD) may present with extrarenal cyst formation, sometimes involving the pancreas. Large enough, these cysts may cause pancreatitis in ADPKD patients.Case presentationHerein, we present a case of a 12-year-old Caucasian girl with recurrent pancreatitis with no identifiable traumatic, metabolic, infectious, drug, or immunologic causes. Structural anomalies of the pancreas, including cysts, were ruled out by imaging. However, bilateral cystic kidneys were found as an incidental finding. Her family history was negative for pancreatitis, but positive for polycystic kidney disease. Molecular analysis of ADPKD-causing mutations revealed a novel c.9659C>A (p.Ser3220*) mutation in the PKD1 gene confirming the clinical suspicion of ADPKD. Although CP may rarely occur as an extrarenal manifestation of ADPKD with pancreatic cysts, it is unusual in their absence. Thus, molecular analysis of pancreatitis susceptibility genes was performed and a homozygous pathologic c.180C>T (p.G60=) variant of the CTRC gene, known to increase the risk of CP, was confirmed.ConclusionThis is the first reported case of a pediatric patient with coincidence of genetically determined CP and ADPKD. Occurrence of pancreatitis in children with ADPKD without pancreatic cysts warrants further investigation of CP causing mutations.     

Cardiovascular polycystins: insights from autosomal dominant polycystic kidney disease and transgenic animal models

Mutations in the PKD1 and PKD2 polycystin genes are responsible for autosomal dominant polycystic kidney disease (ADPKD), one of the most prevalent genetic kidney disorders. ADPKD is a multisystem disease characterized by the formation of numerous fluid-filled cysts in the kidneys, the pancreas, and the liver. Moreover, major cardiovascular manifestations are common complications in ADPKD. Intracranial aneurysms and arterial hypertension are among the leading causes of mortality in this disease. In the present review, we summarize our current understanding of the role of polycystins in the development, maintenance, and function of the cardiovascular system.     

Specific association of the gene product of PKD2 with the TRPC1 channel

The function(s) of the genes (PKD1 and PKD2) responsible for the majority of cases of autosomal dominant polycystic kidney disease is unknown. While PKD1 encodes a large integral membrane protein containing several structural motifs found in known proteins involved in cell-cell or cell-matrix interactions, PKD2 has homology to PKD1 and the major subunit of the voltage-activated Ca2+ channels. We now describe sequence homology between PKD2 and various members of the mammalian transient receptor potential channel (TRPC) proteins, thought to be activated by G protein-coupled receptor activation and/or depletion of internal Ca2+ stores. We show that PKD2 can directly associate with TRPC1 but not TRPC3 in transfected cells and in vitro. This association is mediated by two distinct domains in PKD2. One domain involves a minimal region of 73 amino acids in the C-terminal cytoplasmic tail of PKD2 shown previously to constitute an interacting domain with PKD1. However, distinct residues within this region mediate specific interactions with TRPC1 or PKD1. The C-terminal domain is sufficient but not necessary for the PKD2-TRPC1 association. A more N-terminal domain located within transmembrane segments S2 and S5, including a putative pore helical region between S5 and S6, is also responsible for the association. Given the ability of the TRPC to form functional homo- and heteromultimeric complexes, these data provide evidence that PKD2 may be functionally related to TRPC proteins and suggest a possible role of PKD2 in modulating Ca2+ entry in response to G protein-coupled receptor activation and/or store depletion.     

Pathogenicity of missense and splice site mutations in hMSH2 and hMLH1 mismatch repair genes: implications for genetic testing

BACKGROUND: In hereditary non-polyposis colorectal cancer, over 90% of the identified mutations are in two genes, hMSH2 and hMLH1. A large proportion of the mutations detected in these genes are of the missense type which may be either deleterious mutations or harmless polymorphisms. AIM: To investigate whether nine missense and one splice site mutation of hMLH1 and hMSH2, in 10 kindreds with a familial history of colorectal cancer or young age of onset, could be interpreted as pathogenic. METHODS: Clinical and genetic characteristics were collected: (i) evolutionary conservation of the codon involved; (ii) type of amino acid change; (iii) occurrence of mutation in healthy controls; (iv) cosegregation of mutation with disease phenotype; (v) functional consequences of gene variant; and (vi) microssatellite instability and immunoexpression of hMSH2 and hMLH1 analysis. RESULTS: Seven different missense and one splice site mutation were identified. Only 1/8 was found in the control group, 2/7 occurred in conserved residues, and 5/7 resulted in non-conservative changes. Functional studies were available for only 2/8 mutations. Segregation of the missense variant with disease phenotype was observed in three kindreds. CONCLUSION: In the majority of families included, there was no definitive evidence that the missense or splice site alterations were causally associated with an increased risk of developing colorectal cancer. Until further evidence is available, these mutational events should be regarded and interpreted carefully and genetic diagnosis should not be offered to these kindreds.     

Molecular Diagnostics in Autosomal Dominant Polycystic Kidney Disease: Utility and Limitations

Background and objectives: Gene-based mutation screening is now available and has the potential to provide diagnostic confirmation or exclusion of autosomal dominant polycystic kidney disease. This study illustrates its utility and limitations in the clinical setting.Design, setting, participants, & measurements: Using a molecular diagnostic service, genomic DNA of one affected individual from each study family was screened for pathologic PKD1 and PKD2 mutations. Bidirectional sequencing was performed to identify sequence variants in all exons and splice junctions of both genes and to confirm the specific mutations in other family members. In two multiplex families, microsatellite markers were genotyped at both PDK1 and PKD2 loci, and pair-wise and multipoint linkage analysis was performed.Results: Three of five probands studied were referred for assessment of renal cystic disease without a family history of autosomal dominant polycystic kidney disease, and two others were younger at-risk members of families with autosomal dominant polycystic kidney disease being evaluated as living-related kidney donors. Gene-based mutation screening identified pathogenic mutations that provided confirmation or exclusion of disease in three probands, but in the other two, only unclassified variants were identified. In one proband in which mutation screening was indeterminate, DNA linkage studies provided strong evidence for disease exclusion.Conclusions: Gene-based mutation screening or DNA linkage analysis should be considered in individuals in whom the diagnosis of autosomal dominant polycystic kidney disease is uncertain because of a lack of family history or equivocal imaging results and in younger at-risk individuals who are being evaluated as living-related kidney donors.Autosomal dominant polycystic kidney disease (ADPKD) is the most common hereditary kidney disease worldwide affecting one in 500 to 1000 live births (1,2). It is characterized by focal and sporadic development and progressive enlargement of renal cysts, leading to ESRD in late middle age. Typically, only a few renal cysts are detected in most affected individuals before 30 yr of age; however, by the fifth decade of life, hundreds to thousands of renal cysts will be found in the majority of patients. Overall, it accounts for 5 to 8% of ESRD in developed countries (1,2). ADPKD is a systemic disorder associated with multiple extrarenal complications, such as cysts in nonrenal organs, valvular heart disease, colonic diverticula, inguinal hernias, and intracranial arterial aneurysms. It is genetically heterogeneous, with most cases arising from mutations in PKD1 (MIM 601313) and PKD2 (MIM 173910), located on chromosome 16p13.3 and 4q21–23, respectively (1–4). In a linkage-characterized European sample, PKD1 accounts for approximately 85% of the cases, whereas PKD2 accounts for most of the remainder (3,4). Although the clinical manifestations of the two gene types overlap completely, a strong locus effect is evident with more severe renal disease in PKD1 than PKD2 (median age at ESRD 54 versus 74, respectively) (5). In addition, both environmental and genetic modifiers have been implicated to account for the significant intrafamilial renal disease variability observed (6–8), and a mild allelic effect has been suggested for PKD1 but not PKD2 (7,8).PKD1 is a large gene consisting of 46 exons with an open reading frame of approximately 13 kb and is predicted to encode a protein of 4302 amino acids. Its entire 5′ region up to exon 33 has been duplicated six times more proximally on chromosome 16p, and the presence of these highly homologous pseudogenes has made genetic analysis of PKD1 difficult (1,2). Recent availability of protocols for long-range and locus-specific amplification of PKD1 has enabled the complete mutation screening of this complex gene (9–11). By contrast, PKD2 is a single-copy gene that consists of 15 exons with an open reading frame of approximately 3 kb and is predicted to encode a protein of 968 amino acids (1,2). Marked allelic heterogeneity is evident for ADPKD, with more than 200 different PKD1 and more than 50 different PKD2 mutations reported to date (2,9–11). The majority of these mutations are unique and scattered throughout both genes. Most of them are also predicted to be protein truncating (as a result of frame-shift deletion/insertion, nonsense changes, or splice defects), although a significant number of unclassified variants (UCV; e.g., in-frame deletions, missense changes) have been reported (9–11). Despite sequencing of all of the coding regions and exon-intron splice junctions in both genes only 45 to 63% of pathogenic mutations could be identified in three large clinical series (9–11).The diagnosis of ADPKD is generally straightforward when affected individuals present with a positive family history and enlarged kidneys with multiple cysts (12). Renal ultrasound is a sensitive method for this purpose, and age-dependant criteria based on cyst number have been derived for individuals who are born with 50% risk for PKD1 (13); however, because cyst formation is an age-dependent process, the false-negative rate of ultrasound-based diagnosis is higher in younger at-risk individuals or in those who are affected by PKD2, which is associated with later onset disease (14). Equivocal imaging results can be a source of diagnostic uncertainty in the clinic because the underlying gene type for most patients is unknown. In addition, renal cystic disease without a family history of ADPKD and evaluation of younger at-risk individuals as living-related kidney donors are clinical scenarios that often pose diagnostic challenges (12). Using a case series, we illustrate the utility and limitations of molecular diagnostics for ADPKD in the clinical setting.     

Chemical modifier screen identifies HDAC inhibitors as suppressors of PKD models

Polycystic kidney disease (PKD) is a common human genetic disease with severe medical consequences. Although it is appreciated that the cilium plays a central role in PKD, the underlying mechanism for PKD remains poorly understood and no effective treatment is available. In zebrafish, kidney cyst formation is closely associated with laterality defects and body curvature. To discover potential drug candidates and dissect signaling pathways that interact with ciliary signals, we performed a chemical modifier screen for the two phenotypes using zebrafish pkd2^hi4166 and ift172^hi2211 models. pkd2 is a causal gene for autosomal dominant PKD and ift172 is essential for building and maintaining the cilium. We identified trichostatin A (TSA), a pan-HDAC (histone deacetylase) inhibitor, as a compound that affected both body curvature and laterality. Further analysis verified that TSA inhibited cyst formation in pkd2 knockdown animals. Moreover, we demonstrated that inhibiting class I HDACs, either by valproic acid (VPA), a class I specific HDAC inhibitor structurally unrelated to TSA, or by knocking down hdac1, suppressed kidney cyst formation and body curvature caused by pkd2 deficiency. Finally, we show that VPA was able to reduce the progression of cyst formation and slow the decline of kidney function in a mouse ADPKD model. Together, these data suggest body curvature may be used as a surrogate marker for kidney cyst formation in large-scale high-throughput screens in zebrafish. More importantly, our results also reveal a critical role for HDACs in PKD pathogenesis and point to HDAC inhibitors as drug candidates for PKD treatment.     

Inhibiting the HSP90 chaperone slows cyst growth in a mouse model of autosomal dominant polycystic kidney disease

Autosomal dominant polycystic kidney disease (ADPKD) is a progressive genetic syndrome with an incidence of 1:500 in the population, arising from inherited mutations in the genes for polycystic kidney disease 1 (PKD1) or polycystic kidney disease 2 (PKD2). Typical onset is in middle age, with gradual replacement of renal tissue with thousands of fluid-filled cysts, resulting in end-stage renal disease requiring dialysis or kidney transplantation. There currently are no approved therapies to slow or cure ADPKD. Mutations in the PKD1 and PKD2 genes abnormally activate multiple signaling proteins and pathways regulating cell proliferation, many of which we observe, through network construction, to be regulated by heat shock protein 90 (HSP90). Inhibiting HSP90 with a small molecule, STA-2842, induces the degradation of many ADPKD-relevant HSP90 client proteins in Pkd1^−/− primary kidney cells and in vivo. Using a conditional Cre-mediated mouse model to inactivate Pkd1 in vivo, we find that weekly administration of STA-2842 over 10 wk significantly reduces initial formation of renal cysts and kidney growth and slows the progression of these phenotypes in mice with preexisting cysts. These improved disease phenotypes are accompanied by improved indicators of kidney function and reduced expression and activity of HSP90 clients and their effectors, with the degree of inhibition correlating with cystic expansion in individual animals. Pharmacokinetic analysis indicates that HSP90 is overexpressed and HSP90 inhibitors are selectively retained in cystic versus normal kidney tissue, analogous to the situation observed in solid tumors. These results provide an initial justification for evaluating HSP90 inhibitors as therapeutic agents for ADPKD.     

Isolated polycystic liver disease as a distinct genetic disease,unlinked to polycystic kidney disease 1 and polycystic kidney disease 2

Polycystic liver disease (PLD) is proven to occur either sporadically or in association with autosomal dominant polycystic kidney disease (ADPKD), whereas the existence of an isolated (i.e., without any kidney cyst) familial form is disputed. We describe a family with definitely isolated PLD transmitted through three generations and exclude the linkage of the disease to the genetic markers of PKD1 and PKD2, the two main loci responsible for ADPKD. These findings strongly support the existence of PLD as a genetic disease distinct from the known forms of ADPKD. (Hepatology 1996 Feb;23(2):249-52)     

Mutant polycystin-2 induces proliferation in primary rat tubular epithelial cells in a STAT-1/p21-independent fashion accompanied instead by alterations in expression of p57KIP2 and Cdk2

Background

Autosomal Dominant Polycystic Kidney Disease (ADPKD) is characterized by the formation of multiple fluid-filled cysts that destroy the kidney architecture resulting in end-stage renal failure. Mutations in genes PKD1 and PKD2 account for nearly all cases of ADPKD. Increased cell proliferation is one of the key features of the disease. Several studies indicated that polycystin-1 regulates cellular proliferation through various signaling pathways, but little is known about the role played by polycystin-2, the product of PKD2. Recently, it was reported that as with polycystin-1, polycystin-2 can act as a negative regulator of cell growth by modulating the levels of the cyclin-dependent kinase inhibitor, p21 and the activity of the cyclin-dependent kinase 2, Cdk2.

Methods

Here we utilized different kidney cell-lines expressing wild-type and mutant PKD2 as well as primary tubular epithelial cells isolated from a PKD transgenic rat to further explore the contribution of the p21/Cdk2 pathway in ADPKD proliferation.

Results

Surprisingly, over-expression of wild-type PKD2 in renal cell lines failed to inactivate Cdk2 and consequently had no effect on cell proliferation. On the other hand, expression of mutated PKD2 augmented proliferation only in the primary tubular epithelial cells of a rat model but this was independent of the STAT-1/p21 pathway. On the contrary, multiple approaches revealed unequivocally that expression of the cyclin-dependent kinase inhibitor, p57^KIP2, is downregulated, while p21 remains unchanged. This p57 reduction is accompanied by an increase in Cdk2 levels.

Conclusion

Our results indicate the probable involvement of p57^KIP2 on epithelial cell proliferation in ADPKD implicating a new mechanism for mutant polycystin-2 induced proliferation. Most importantly, contrary to previous studies, abnormal proliferation in cells expressing mutant polycystin-2 appears to be independent of STAT-1/p21.     

The polycystic kidney disease protein PKD2 interacts with Hax-1, a protein associated with the actin cytoskeleton

Despite the recent positional cloning of the PKD1 and PKD2 genes, which are mutated in the great majority of patients with autosomal-dominant polycystic kidney disease (PKD), the pathogenic mechanism for cyst formation is still unclear. The finding, that the PKD1 and PKD2 proteins interact with each other through their COOH termini, suggests that both proteins are part of the same protein complex or signal transduction pathway. Using a yeast two-hybrid screen with the PKD2 protein, we isolated the PKD2-interacting protein Hax-1. The specificity of the interaction was demonstrated by the fact that PKD2L, a protein closely related to PKD2, failed to interact with Hax-1. Immunofluorescence experiments showed that in most cells PKD2 and Hax-1 colocalized in the cell body, but in some cells PKD2 and Hax-1 also were sorted into cellular processes and lamellipodia. Furthermore we demonstrated an association between Hax-1 and the F-actin-binding protein cortactin, which suggests a link between PKD2 and the actin cytoskeleton. We speculate that PKD2 is involved in the formation of cell-matrix contacts, which are dysfunctional without a wild-type PKD2 protein, thus leading to cystic enlargement of tubular structures in the kidney, liver, and pancreas.     

Evidence for Pathogenicity of Atypical Splice Mutations in Autosomal Dominant Polycystic Kidney Disease

Background and objectives: Mutation-based molecular diagnostics of autosomal dominant polycystic kidney disease (ADPKD) is complicated by locus and allelic heterogeneity, large multi-exon gene structure and duplication in PKD1, and a high level of unclassified variants. Comprehensive screening of PKD1 and PKD2 by two recent studies have shown that atypical splice mutations account for 3.5% to 5% of ADPKD. We evaluated the role of bioinformatic prediction of atypical splice mutations and determined the pathogenicity of an atypical PKD2 splice variant from a multiplex ADPKD (TOR101) family.Design, setting, participants, & measurements: Using PubMed, we identified 17 atypical PKD1 and PKD2 splice mutations. We found that bioinformatics analysis was often useful for evaluating the pathogenicity of these mutations, although RT-PCR is needed to provide the definitive proof.Results: Sequencing of both PKD1 and PKD2 in an affected subject of TOR101 failed to identify a definite mutation, but revealed several UCVs, including an atypical PKD2 splice variant. Linkage analysis with microsatellite markers indicated that TOR101 was PKD2-linked and IVS8 + 5G→A was shown to cosegregate only with affected subjects. RT-PCR of leukocyte mRNA from an affected subject using primers from exons 7 and 9 revealed six splice variants that resulted from activation of different combinations of donor and acceptor cryptic splice sites, all terminating with premature stop codons.Conclusions: The data provide strong evidence that IVS8 + 5G→A is a pathogenic mutation for PKD2. This case highlights the importance of functional analysis of UCVs.Autosomal dominant polycystic kidney disease (ADPKD) is the most common hereditary kidney disorder worldwide, affecting approximately one in 500 live births. It is characterized by focal development and progressive enlargement of renal cysts, leading to end-stage renal disease (ESRD) in late middle age. Typically, only a few renal cysts are detected in most affected subjects before 30 yr of age. However, by the fifth decade of life, hundreds to thousands of renal cysts are found in most patients. Overall, ADPKD accounts for 5% to 8% of end-stage renal disease (ESRD) in developed countries (1,2). Extrarenal complications of ADPKD are variable and include inguinal hernias, colonic diverticulae, valvular heart disease, and intracranial arterial aneurysms (1).Mutations of two genes, PKD1 (MIM 601313) and PKD2 (MIM 173910), account for approximately 85% and 15% of all cases of ADPKD in linkage-characterized European populations (3,4). Although the clinical manifestations of PKD1 and PKD2 overlap completely, a strong locus effect on renal disease severity is evident with more severe renal disease in PKD1 than PKD2 (median age at ESRD: 54 yr versus 74, respectively) (5). PKD1 is a large gene consisting of 46 exons with an open reading frame of approximately 13 kb and is predicted to encode a protein of 4302 amino acids. Its entire 5′ region up to exon 33 has been duplicated six times proximally on chromosome 16p, and the presence of these highly homologous pseudogenes has made genetic analysis of PKD1 difficult (1,2). Recent availability of protocols for long-range and locus-specific amplification of PKD1 has enabled the complete mutation screening of this complex gene (6–9). In contrast, PKD2 is a single-copy gene consisting of 15 exons with an open reading frame of approximately 3 kb and is predicted to encode a protein of 968 amino acids (1,2).The diagnosis of ADPKD is straightforward in affected subjects with a positive family history and enlarged kidneys with multiple cysts (6). Renal ultrasound is a useful method for this purpose, and age-dependant criteria based on cyst number have been derived for subjects born with 50% risk of PKD1 or PKD2 (6,10). However, ultrasound diagnosis of ADPKD in younger at-risk subjects with equivocal or negative findings and in subjects affected by PKD2 or de novo disease remains a challenge (6). For these reasons, molecular screening is a useful tool in the clinical setting. However, marked allelic heterogeneity is evident, with over 200 different PKD1 and over 50 different PKD2 mutations reported to date (2,6–9,11–13). The majority of these mutations are unique and scattered throughout both genes. Although the majority of these mutations are predicted to be protein truncating (frame-shift deletion/insertion, nonsense or canonical splice changes), a large number of unclassified variants (UCVs; in-frame deletions, mis-sense and atypical splice changes) has also been reported (7–9). Comprehensive screening of both PKD1 and PKD2 by two recent studies identified definitive and probable mutations in 42% to 63% and 26% to 37% of patients, respectively (8,9). These two studies also reported that atypical splice mutations account for approximately 3.5% to 5% of ADPKD (8,9). In the current study, we performed and evaluated the utility of bioinformatics analysis on 17 reported atypical PKD1 and PKD2 splice mutations. We also determine the pathogenicity of an atypical splice variant found in a family affected by PKD2 and highlight the importance of functional analysis of UCVs in molecular diagnostic testing.