Polycystic kidney disease PKD2 gene and uses thereof

Abstract

The present invention provides a purified and isolated wild type PKD2 gene, as well as mutated forms of this gene. The present invention also provides one or more single-stranded nucleic acid probes which specifically hybridize to the wild type PKD2 gene or the mutated PKD2 gene, and mixtures thereof, which may be formulated in kits, and used in the diagnosis of ADPKD associated with the mutated PKD2 gene. The present invention also provides a method for diagnosing ADPKD caused by a mutated PKD2 gene, as well as a method for treating autosomal dominant polycystic kidney disease caused by a mutated PKD2 gene.

Description

BACKGROUND OF THE INVENTION

This invention is based upon the discovery by the inventors of the PKD2 gene associated with Autosomal Dominant Polycystic Kidney Disease (“ADPKD”), the “PKD2 gene” or “PKD2”, and a novel protein encoded by this gene. The discovery of the PKD2 gene and the protein encoded by the gene will have important implications in the diagnosis and treatment of ADPKD caused by defects in the PKD2 gene.

ADPKD is a genetically heterogeneous disorder that affects approximately 500,000 Americans and five million individuals world wide, and accounts for 8 to 10% of all end stage renal disease (ESRD) worldwide (Gabow, P. A. N. Eng. J. Med. 329:332 (1993)). Its principal clinical manifestation is bilateral renal cysts that result in chronic renal failure in about 45% of affected individuals by age 60 (Gabow, P. A., supra). Hypertension and liver cysts are common, and the involvement of other organ systems (Gabow, P. A., et al. Kidney Int. 38:1177 (1990); Chapman, A. B., et al. N. Eng. J. Med. 327:916 (1992); Hossack, K. F., et al. N. Eng. J. Med. 319:907 (1988); Torres, V. E., et al. Am. J. Kidney Dis. 22:513 (1993); Huston, J., et al. J. Am. Soc. Nephrol. 3:1871 (1993); Somlo, S., et al. J. Am. Soc. Nephrol. 4:1371 (1993)) lends support to the view that polycystic kidney disease is a systemic disorder (Gabow, P. A., supra).

To date, most forms of ADPKD have been associated with two genes, PKD1 and PKD2. The full genomic structure and cDNA sequence for the PKD1 gene has been identified (The International Polycystic Kidney Disease Consortium, Cell 81:289 (1995); The American PKD1 Consortium, Hum. Mol. Genet. 4:575 (1995)). Mutations in the PKD1 gene are suspected of causing 80-90% of all cases of ADPKD. The PKD2 gene has been localized on chromosome 4q21-23 and accounts for approximately 15% of affected families (Kimberling, W. J., et al. Genomics 18:467 (1993); Peters, D. J. M. and L. A. Sandkuijl Contrib. Nephrol. 97:128 (1992)). Prior to the present invention, however, the PKD2 gene had not been identified.

SUMMARY OF THE INVENTION

The present invention provides a purified and isolated wild type PKD2 gene, as well as mutated forms of this gene. The present invention also provides one or more single-stranded nucleic acid probes which specifically hybridize to the wild type PKD2 gene or the mutated PKD2 gene, and mixtures thereof, which may be formulated in kits, and used in the diagnosis of ADPKD associated with the mutated PKD2 gene.

The present invention also provides a vector comprising nucleic acid encoding an active PKD2 protein, a cell stably transformed with this vector, as well as a method for producing recombinant, active PKD2 protein. A purified, active PKD2 protein is also provided by the present invention. In addition, the present invention provides an antibody immunoreactive with a wild type PKD2 protein, as well as an antibody immunoreactive with a mutant PKD2 protein, which may be formulated in kits, and used in the diagnosis of ADPKD associated with the mutated PKD2 gene.

The present invention further provides a method for diagnosing ADPKD caused by a mutated PKD2 gene in an adult subject suspected of having the disease comprising detecting the presence of a mutated PKD2 gene in nucleic acid of the subject. The present invention still further provides a method for treating ADPKD caused by a mutated PKD2 gene in a subject in need of such treatment comprising the delivery and expression of a functional PKD2 gene into a sufficient number of cells of the subject to treat the disease. A stem cell which expresses the PKD2 gene introduced therein through viral transduction, homologous recombination or transfection is also provided by the invention.

In addition, the present invention provides a recombinant viral vector for treating a defect in the PKD2 gene in a target cell comprising (a) the nucleic acid of or corresponding to at least a portion of the genome of a virus, which portion is capable of directing the infection of the target cell, and (b) a PKD2 gene operably linked to the viral nucleic acid and capable of being expressed as a functional gene product in the target cell.

Finally, the present invention provides a vector and an embryonic stem cell each of which comprises a mutated PKD2 gene, a non-human, transgenic animal whose germ and somatic cells contain a mutated PKD2 gene sequence introduced into said animal, or an ancestor thereof, at an embryonic stage, as well as a method for producing the non-human, transgenic animal.

Additional objects of the invention will be apparent from the description which follows.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A represents the subset of STSs from the high density map of the PKD2 region showing polymorphic loci flanking the interval. JSTG3 and AICA1 are two of nine microsatellite markers in this region developed previously. SPP1 (osteopontin, STS4-1078) and D4S1171 were used to screen the P1 library as described in Materials and Methods. Other sources of STSs include published linkage maps and genome center databases. cen, centromere; tel telomere. Distances are in Morgans along chromosome 4.

FIG. 1B shows representative mega-YACs (Cohen, D., et al. Nature 366:698 (1993)), and their STS content, forming a contig around the PKD2 region.

FIG. 1C shows the minimum tiling path of the cosmid and P1 contig in the PKD2 region. Clone names beginning with “c” and “p” refer to cosmid and P1 clones, respectively; addresses are from the original arrayed libraries. The clones containing JSTG3 and AICA1 are shown; a single gap of <40 kb is indicated by the arrow.

FIG. 1D shows the detail of the portion of the contig containing the PKD2 candidate gene, cTM-4.

FIG. 1E shows overlapping map of nine cDNA clones for cTM-4 and a composite schematic at the bottom. Clones K1-1 and K1-5 are from the adult kidney library; clones yj63h09 and yc93g07 were identified by GenBank searching and are from the normalized infant brain library (Soares, M. B., et al. Proc. Natl. Acad. Sci. USA 91: 9228 (1994)); all other clones are from the fetal brain library. Shaded areas represent chimeric portions of clones.

FIG. 2 represents expression of the PKD2 candidate gene. Insert from cTM-4B3-3 ( FIG. 1E ) was used as a hybridization probe on mRNA blots containing human tissues (Clonetech, Palo Alto, Calif.). Hybridization was performed without pre-competition and a final wash stringency of 0.5X SSC, 0.1% SDS at 65° C. Tissues in numbered lanes are: (1) heart, (2) brain, (3) placenta, (4) lung, (5) liver, (6) skeletal muscle, (7) kidney, (8) pancreas, (9) spleen, (10) thymus, (11) prostate, (12) testis, (13) ovary, (14) small intestine, (15) colon, (16) leukocytes, (17) fetal brain, (18) fetal lung, (19) fetal liver, (20) fetal kidney. At bottom, β-actin hybridization to the same blots is used to compare relative mRNA loading within each blot.

FIG. 3 depicts the mutations in PKD2 from an analysis of genomic PCR products in three PKD2 families. Left panel shows the results of direct sequencing of genomic PCR products from affected individuals. The arrows denote double peaks, confirmed by sequencing in both directions, indicative of heterozygosity at that nucleotide. Each of the mutant alleles results in a premature stop codon. The right panel demonstrates segregation of the mutated allele with the disease phenotype. In families 97 and 1605, the affected alleles are not digested by Bsr I and Taq I, respectively, since the restriction sites are lost by mutation. Family 1601 shows segregation of the SSCA variant, indicated by the arrow, with the disease phenotype. For each family, only portions of more extensive pedigrees are shown. Filled symbols, affected individuals. Open symbols, unaffected individuals. M, 100 bp ladder.

FIG. 4A depicts the deduced amino acid sequence of PKD2 (SEQ ID NO:1) (cTM-4) (GenBank accession: gblU50928) in alignment with PKD1 (SEQ ID NO:2) (gb|U24497), the C. elegans homolog of PKD1 (SEQ ID NO:3) (ZK945.9; swiss|Q09624) and VACCα1E-1 (SEQ ID NO:4) (pir|B54972) using BESTFIT (Program Manual for the Wisconsin Package, Version 8, September 1994): identity to cTM-4, |; similarity to cTM-4, :. Numbers in parentheses refer to amino acids in respective sequences. Putative transmembrane domains, tm1 to tm6. Predicted N-glycosylation sites, *. Potential phosphorylation sites with strong consensus sequences: protein kinase C, +; cGMP dependent kinase, open square (Ser 826 is also consistent with a protein kinase A site); casein kinase, open circle. The sites of the nonsense mutations ( FIG. 3 ) are indicated by arrows labeled with the respective family numbers. The EF-hand domain is indicated by the dashed line.

FIG. 4B shows alignment of the EF-hand domain (SEQ ID NO:5) with the EF-hand test sequence. The residues E, G, I, and E, the latter being a Ca 2+ coordination vertex, are the expected residues at the indicated positions in the EF-hand. Positions indicated as “n” are expected to have hydrophobic amino acids (L, I, V, F, M); those denoted with * should be oxygen-containing amino acids (D, N, E, Q, S, T) comprising the remainder of coordination vertices for Ca 2+ binding; the −Y vertex can be any amino acid. The Leu (L) in PKD2 in place of the Ile (I) is likely a permissible substitution; PKD2 has Gln (Q) in place of the consensus Gly (G) as is the case with EF-hand domains in the α1 Na + channels.

FIGS. 5A-G represents the nucleotide sequence (SEQ ID NO:6) of the PKD2 gene and the deduced amino acid sequence (SEQ ID NO:7) for PKD2.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a purified and isolated wild type PKD2 nucleic acid, as well as mutated forms of this nucleic acid. As used herein, the “wild type PKD2 nucleic acid” is the normal form of the gene which expresses an enzymatically active gene product, and includes degenerate forms. The “mutated PKD2 nucleic acid” is the mutated form of the normal PKD2 gene, which contains one or more deletion, insertion, point or rearrangement mutations, or a combination thereof, that may render the gene product expressed by the mutated PKD2 gene nonfunctional or nonexistent. As used herein, “nucleic acid” may be genomic DNA, cDNA or RNA.

The present invention also provides single-stranded nucleic acid probes and mixtures thereof for use in diagnosing ADPKD caused by a mutated PKD2 gene. The nucleic acid probes may be DNA, cDNA, or RNA, and may be prepared from the mutated and/or wild type PKD2 gene. The probes may be the full length sequence of PKD2 gene, or fragments thereof. Typical probes are 12 to 40 nucleotides in length. Generally, the probes are complementary to the PKD2 gene coding sequences, although probes to introns are also contemplated. The probes may be synthesized using an oligonucleotide synthesizer such as Applied Biosystems Model 392 DNA/RNA synthesizer, and may be labeled with a detectable marker such as a fluorescence, enzyme or radiolabeled markers including 32 P and biotin, and the like. Combinations of two or more labelled probes corresponding to different regions of the PKD2 gene also may be included in kits to allow for the detection and/or analysis of the PKD2 gene by hybridization.

The present invention also provides a vector comprising nucleic acid encoding an active PKD2 protein, as well as a cell stably transformed with the vector. The vector may be any plasmid, viral-derived nucleic acid, lytic bacteriophage derived from phage lambda, cosmid, filamentous single-stranded bacteriophage such as M13, and the like, for cloning nucleic acid or introducing the nucleic acid into a cell for expression. The cell may be eukaryotic or prokaryotic. Suitable host cells include but are not limited to bacterial cells such as E. coli, Bacillus subtilis, Agrobacterium tumefaciens, Bacillus subtilis, Agrobacterium tumefaciens, Bacillus megaterium , eukaryotic cells such as Pichia pastoris, Chlamydomonas reinhardtii, Cryptococcus neoformans, Neurospora crassa, Podospora anserina, Saccharomyces cerevisiae, Saccharomyces pombe, Uncinula necator , cultured insect cells, cultured chicken fibroblasts, cultured hamster cells, cultured human cells such as HT1080, MCF7, 143B and cultured mouse cells such as EL4 and NIH3T3 cells. Such expression systems may be used to produce a recombinant, active PKD2 protein by culturing a cell transformed with a vector comprising a nucleic acid encoding an active PKD2 protein, and recovering PKD2 protein from the culture.

The present invention also provides a purified active PKD2 protein. The protein may be the wild type protein or an analogue thereof. As used herein, “analogue” means functional variants of the wild type protein, and includes PKD2 proteins isolated from mammalian sources other than human, as well as functional variants thereof. The protein also may be isolated from native cells or recombinantly produced.

The present invention also provides antibodies immunoreactive with the protein expressed by the wild type PKD2 gene (and analogues thereof), as well as antibodies immunoreactive with the protein expressed by the mutated PKD2 gene. The antibodies may be polyclonal or monoclonal and are produced by standard techniques. The antibodies may be labeled with standard detectable markers (e.g. chemiluminescent detection systems and radioactive labels such as 125 I) for detecting the wild type and mutated PKD2 genes. The antibodies also may be presented in kits with detectable labels and other reagents and buffers for such detection.

The present invention also provides a method for diagnosing ADPKD in a subject comprising detecting the presence of a mutated PKD2 gene in nucleic acid of the subject. The method may be used to determine whether persons in the population at large have ADPKD, for identifying persons at risk in developing the disease, i.e. relatives of persons with ADPKD, as well as for confirming diagnosis of ADPKD. The method also is useful for diagnosing ADPKD before clinical manifestations of the disease, i.e. the formation of cysts. Accordingly, as used herein, “subject” may be an embryo, fetus, newborn, infant or adult.

The presence of the mutated PKD2 gene may be detected by procedures known in the art including but not limited to standard sequencing techniques (e.g. dideoxy chain termination), restriction enzyme digestion analysis, hybridization with one or more probes hybridizable to the mutated and/or wild type PKD2 gene using standard procedures such as Southern blot analysis, polymerase chain reaction using sense and antisense primers prepared from the mutated and/or wild type PKD2 genes, and combinations thereof.

The presence of the mutated PKD2 gene also may be detected by detecting expression of the gene product of the gene. Such expression products include both mRNA as well as the protein product itself. mRNA expression may be detected by standard sequencing techniques, hybridization with one or more probes hybridizable to the mutated and/or wild type PKD2 mRNA using standard procedures such as Northern blot analyses, dot and slot hybridization, S1 nuclease assay, or ribonuclease protection assays, polymerase chain reaction using sense and antisense primers prepared from the mutated and/or wild type PKD2 genes, and combinations thereof. The protein may be detected using antibodies to the protein expressed by the mutated PKD2 gene and/or the wild type PKD2 gene by procedures known in the art including but not limited to immunoblotting, immunoprecipitation, solid phase radioimmunoassay (e.g. competition RIAs, immobilized antigen or antibody RIAs, or double antibody RIAs), enzyme-linked immunoabsorbent assay, and the like.

The present invention also provides a method for treating ADPKD caused by a mutated PKD2 gene in a subject in need of such treatment comprising the delivery and expression of a functional PKD2 gene into a sufficient number of cells of the subject, preferably bone marrow stem cells, to treat ADPKD in the subject. As used herein, “functional PKD2 gene” is a gene which when incorporated into a cell's nucleic acid expresses a functional gene product, and includes the wild type PKD2 gene as well as variations thereof. The delivery and expression of the functional PKD2 gene may be accomplished by introducing the functional PKD2 gene into the cells or by correcting the mutation(s) in the subject's PKD2 gene.

The functional PKD2 gene may be delivered into the subject's cells by a number of procedures known to one skilled in the art, e.g. electroporation, DEAE dextran, cationic liposome fusion (using both monocationic and polycationic lipids), protoplast fusion, DNA coated microprojectile bombardment, injection with recombinant replication-defective retroviruses, homologous recombination, and the like. Accordingly, a stem cell which expresses the PKD2 gene introduced therein through viral transduction, homologous recombination, or transfection is also provided by the present invention.

The present invention also provides a recombinant viral vector for treating a defect in the PKD2 gene in a target cell comprising (a) the nucleic acid of or corresponding to at least a portion of the genome of a virus, which portion is capable of directing the infection of the target cell, and (b) a functional PKD2 gene operably linked to the viral nucleic acid and capable of being expressed as a functional gene product in the target cell. The recombinant viral vectors of the present invention may be derived from a variety of viral nucleic acids known to one skilled in the art, e.g. the genomes of HSV, adenovirus, adeno-associated virus, Semiliki Forest virus, vaccinia virus, and other retroviruses or DNA viruses.

The present invention also provides a vector for use in preparing a non-human, transgenic animal comprising a mutated PKD2 gene which is capable of introducing the mutated PKD2 gene in at least some embryonic cells to which the vector is introduced, an embryonic stem cell comprising a mutated PKD2 gene which has been integrated into the cell following transduction with the vector above, as well as a non-human transgenic animal of ADPKD which would be useful for studying ADPKD. The mutated PKD2 gene may be integrated into the germ line of a non-human animal such as a mouse, rat, goat, sheep or other non-human species in order to obtain a transgenic animal model by methods known in the art (see Alberts, B., et al. Molecular Biology of the Cell, 2d. Garland Publ. Inc., New York and London, pp. 267-269 (1989)). For example, nucleic acid encoding the mutated PKD2 protein can be inserted into the genome of a replication-defective virus such as HSV or a retrovirus or transposen and the resultant construct injected into embyronic stem cells. Alternatively, the transgenic animal may be made by injecting nucleic acid into the male pronucleus of a fertilized egg of a nonhuman animal, transplanting the “transgenic embryo” into a pseudopregnant female and then analyzing offspring for the presence of the injected nucleic acid in their genome.

The present invention is described in the following Experimental Details Section, which is set forth to aid in an understanding of the invention, and should not be construed to limit in any way the invention as defined in the claims which follow thereafter.

EXPERIMENTAL DETAILS SECTION

A. Materials and Methods

Cosmid and P1 Contig Construction. Cosmid and P1 contig construction was guided by the existing YAC contig (Mochizuki, T., et al., unpublished observations; Veldhuisen, B., et al., unpublished observations). Cosmid clones were obtained by hybridization screening of the human chromosome 4-specific cosmid library (Riess, O., et al. Cytogenet. Cell Genet. 65:238 (1994); Ioannou, P. A., et al. Nature Genetics 6:84 (1994)). The probes used for hybridization were: 1) pooled Alu products from mega-YAC 967d1, 2) end sequences from cosmid or P1 clones mapping into the region, and 3) internal restriction fragments from the YAC, P1 and cosmid clones. All hybridization probes were α 32 P-dCTP labeled by standard techniques. Human repetitive sequences were pre-competed with 30-80 μg of C o t-1 DNA using the manufacturer's portocol (Gibco/BRL, Gaithsburg, Md.). Hybridization was carried out in Church-Gilbert buffer. The P1 library (Riess, O., et al., supra; Ioannou, P. A., et al., supra) was screened by PCR from colony pools of each 384-well plate using STS4-1078 (SPP1) and D4S1171. Cosmid and P1 clones mapping into the PKD2 interval were screened for STS content to anchor positive clones onto the YAC contig. Overlap relationships among the clones were established by Eco RI fingerprint analysis and by hybridization.

Screening of cDNA Clones. 6×10 5 plaques of oligo-dT and random primed human fetal brain (Stratagene #936206) and adult kidney (Clonetech #HL3001a) cDNA libraries were plated at a density of 3×10 4 per 150 mm plate and replica lifted onto nylon filter circles. Cosmid and P1 inserts used in library screening were released from vector with Not I and purified from agarose gels. The cumulative length of inserts used as probe in a library screening was <80 kb to maintain adequate signal-to-noise. Insert DNA was labeled and pre-competed with 2 μg sCOS-1 vector in addition to C o t-1 DNA. Positively hybridizing plaques were purified by standard techniques and insert DNA was excised (λZAPII) or subcloned (λgt10).

Identification of Mutations. The mutation in family 1605 was detected initially in RT-PCR template using the cDNA-based primers F11 (SEQ ID NO:8) (5′-GGGCTACCATAAAGCTTTG-3′) and R11 (SEQ ID NO:9) (5′-GTTCATGTTCGATCAGTTCT-3′) (205 bp product) and confirmed in genomic DNA using F11 with intronic primer IR11 (SEQ ID NO:10) (5′-GGGCTAGAAATACTCTTATCACC-3′) (201 bp product). The mutations in families 97 and 1601 were initially detected in genomic DNA using intronic primers IF1C (SEQ ID NO:11) (5′-GCCTCAAGTGTTCCACTGAT-3′) and IR1 (SEQ ID NO:12) (5′-AGGTTTTTCTGGGTAACCCTAG-3′) (362 bp product). Amplifications were performed in standard conditions with hot start. Products were labeled by α 32 P-dCTP incorporation, diluted and denatured in formamide buffer prior to electrophoresis. SSCA was performed according to published protocols (Orita, M., et al. Genomics 5:874 (1989)). Sequencing of purified PCR products was performed with either an ABI 373a or 377 automated sequencing apparatus using cycle sequencing with dye terminator chemistries according to the manufacturer's protocol. The PCR primers were used as sequencing primers and all products were sequenced in both directions. The mutation in family 97 results in the loss of a Bsr I site. Genomic DNA amplified with IF1C and IR1 and digested with Bsr I yields products of 261 and 101 bp in the normal allele. The mutation in family 1605 results in the loss of a Taq I site. Genomic DNA amplified with F11 and IR11 and digested with Taq I yields products of 105 and 96 bp in the normal allele. The SSCA conditions used to demonstrate the mutation in the IF1C-IR1 genomic PCR product in family 1601 were 6% acrylamide (29:1), 1X TBE, on a 20 cm gel run at 14° C. and 100 V for 6 hours.

B. Discussion

The PKD2 genetic interval is flanked by the polymorphic markers D4S231 and D4S414/423 (Kimberling, W. J., et al. Genomics 18:467 (1993); Peters, D. J. M., et al. Nature Genetics 5:359 (1993)). A yeast artificial chromosome (YAC) contig and high density sequence tag site (STS) map of this region was constructed as described above (FIG. 1 ). Genetic studies in affected families using physically ordered polymorphic markers led to several progressive refinements of the PKD2 interval (Mochizuki, T., et al. J. Am. Soc. Nephrol. 5:631a (1994); San Millian, J. L., et al. Am. J. Hum. Genet. 56:248 (1995); Peters, D. J. M., et al. Am. J. Hum. Genet. 57:200a (1995); Constantinou-Deltas, C. D., et al. Hum. Genet. 95:416 (1995)). The closest unambiguous flanking genetic markers are AFMa059xc9 proximally and AICA1 distally ( FIGS. 1A , B). A cosmid- and P1-based (Riess, O., et al. supra; Ioannou, P. A., et al., supra) contig extending over ˜680 kb from AICA1 to the region centromeric to the polymorphic marker JSTG3 was constructed as described above (FIG. 1 C). This contig contains a single gap of less than 40 kb. cDNAs corresponding to genes in this region were isolated using inserts from the genomic clones to screen either a human fetal brain or adult kidney cDNA library as described above. The mapping of the cDNA clones identified was confirmed and the clones were sequenced. These sequences were analyzed to identify open reading frames (ORF) and database searches using the BLAST algorithms (Altschul, S. F., et al. J. Mol. Biol. 215:403 (1990)) were performed.

One group of clones, collectively termed cTM-4, were initially isolated using insert DNA from cosmid c44a9 from the chromosome 4-specific cosmid library as described above (FIG. 1 D). None of the cTM-4 clones have nucleotide level homology to any known genes, although 2 randomly-sequenced cDNA clones were identified (FIG. 1 E). Northern blot hybridization with the cTM-4B3-3 insert ( FIG. 1E ) revealed a ˜5.4 kb transcript expressed in most fetal and adult tissues (FIG. 2 ). cTM-4 is strongly expressed in ovary, fetal and adult kidney, testis, small and large intestine, and fetal lung. Peripheral blood leukocytes was the only tissue tested in which expression was not detected.

Initial database searching using the 6 translated reading frames obtained from the sequence of clone cTM-4B3-3 revealed amino acid level homology with polycystin, the PKD1 gene product (The European Polycystic Kidney Disease Consortium, Cell, 77:881 (1994); The International Polycystic Kidney Disease Consortium, Cell 81:289 (1995); The American PKD1 Consortium, Hum. Mol. Genet. 4:575 (1995); Hughes, J., et al. Nature Genetics 10:151 (1995)). Based on its map location, pattern of expression and the observed homology, the cTM-4 gene was further investigated as a candidate for PKD2. Nine overlapping cDNA clones were completely sequenced in both directions (FIG. 1 E). The 5′ end of the cTM-4 gene contains a pair of genomic Not I sites and the 3′ end extends in the telomeric direction beyond the end of the P1 clone p157n2, into cosmid c44a9 ( FIGS. 1C , 1 D). The gene extends over 68 kb of the genome.

The consensus 5057 bp sequence (GenBank accession: gblU50928) is represented schematically in FIG. 1E. A translation start site with a good Kozak consensus sequence (5′-ACCGCG ATG G-3′) (Kozak, M. Nucleic Acids Res. 15:8125 (1987)) was identified 67 bp from the 5′ end of the K1-1 clone and 61 bases after an in-frame stop codon. It is followed by a 2904 bp ORF followed, in turn, by several in-frame stop codons. The 3′ untranslated region is 2086 bp long and contains a consensus polyadenylation signal.

The DNA sequence and expression profiles of cTM-4 was next analyzed in unrelated affected individuals from families with PKD2 (Kimberling, W. J., et al. N. Eng. J. Med. 319:913 (1988); Kimberling, W. J., et al. Genomics 18:467 (1993); Peters, D. J. M., et al. Nature Genetics 5:359 (1993); Constantinou-Deltas, C. D., et al. Hum. Genet. 95:416 (1995)). Reverse transcribed RNA and genomic DNA templates were used to generate PCR products for, single strand conformational analysis (SSCA) as described above. Genomic PCR products of SSCA variants identified in three families were subjected to direct sequencing. Each affected individual was found to be heterozygous for a single base change that resulted in a nonsense mutation (FIG. 3 ). The mutation in family 97 is a G to A transition in the codon for Trp 380 ( FIGS. 3 , 4 ). The mutations in the Cypriot families 1605 and 1601 are C to T transitions in codons Arg 742 and Gln 405, respectively ( FIGS. 3 , 4 ). Using either the resultant loss of a restriction site in families 97 and 1605, or the SSCA pattern in family 1601, segregation of the mutation with the disease phenotype in each family was demonstrated (FIG. 3 ). Analysis of between 90 and 100 normal chromosomes failed to show the predicted affected allele in any case, making it less likely that these sequence differences represent anonymous polymorphisms. These limited findings do not provide evidence for clustering of mutations in PKD2.

The identification of mutations that disrupt the predicted translation product of cTM-4 and the segregation of these mutations with the ADPKD phenotype in three well characterized PKD2 pedigrees, provide strong evidence that cTM-4 is the PKD2 gene. The putative translation product of the cTM-4 ORF is a 968 amino acid sequence with a calculated molecular mass of 110 kD. Modeling with several hydrophobicity algorithms (Rost, B., et al. Protein Sci. 4:521 (1995); Klein, P., et al. Biochim. Biophys. Acta 815:468 (1985); Kyte, J. and R. F. Doolittle J. Mol. Biol. 157:105 (1982); Engelman, D. M., et al. Annu. Rev. Biophys. Chem. 15:321 (1986)) suggest that cTM-4 is an integral membrane protein with six (range, 5 to 8) membrane spanning domains and intracellular NH 2 - and COOH-termini (Sipos, L. and G. von Heijne Eur. J. Biochem. 213:1333 (1993); Nakashima, H. and K. Nishikawa FEBS Lett. 303:141 (1992); Hartmann, E., et al. Proc. Natl. Acad. Sci. USA 86:5786 (1989)). Of the six highest scoring domains, the fourth transmembrane domain (tm4, FIG. 4 ), produced the lowest scores, but was consistently predicted to be a membrane span by several analyses (Rost, B., et al., supra; Klein, P., et al., supra; Kyte, J. and R. F. Doolittle, supra; Engelman, D. M., et al., supra). The “inside positive” rule (Sipos, L. and G. von Heijne, supra; Nakashima, H. and K. Nishikawa, supra; Hartmann, E., et al., supra) strongly supports the predicted topology. The majority of the N-glycosylation sites, occurring in the segment between tm1 and tm2 (FIG. 4 ), are predicted to be extracellular. In addition, potential phosphorylation sites were identified primarily in the COOH-terminal region, as was a putative EF-hand domain (Kretsinger, R. H. Cold Spring Harb. Symp. on Quant. Biol. 52:499 (1987); Babitch, J. Nature 346:321 (1990)), and this region is predicted to be intracellular (FIG. 4 ). If a stable protein product is produced, the mutations in families 97 and 1601 are expected to result in a product with an intact intracellular NH 2 -terminal domain, first transmembrane domain, and part of the first extracellular loop. The mutation in family 1605 is predicted to result in a product lacking the portion of the intracellular COOH-terminus that contains several phosphorylation sites and the EF-hand domain (FIG. 4 ).

There is ˜25% identity and ˜50% similarity between the putative translation product of PKD2 and ˜450 amino acids of polycystin and its C. elegans homolog, ZK945.9 (FIG. 4 ). There is a comparable degree of similarity with ˜270 residues of the voltage activated Ca 2+ channel α 1E (VACCα1E-1; FIG. 4 ). The similarity between PKD2 and polycystin (and ZK945.9) extends over the region tm1 to tm6 in PKD2 but does not include the NH 2 - and COOH-terminal domains. The corresponding region of polycystin has been predicted to contain four transmembrane segments (Hughes, J., et al. Nature Genetics 10:151 (1995)), three of these corresponding to tm1, tm2 and tm5 in the PKD2 gene product and the fourth localizing between tm5 and tm6 of PKD2. The regions corresponding to tm3 and tm4 of PKD2 were not predicted to be membrane spans in that report (Hughes, et al., supra).

The similarity to VACCα1E-1 (Williams, M. E., et al. J. Biol. Chem. 269:22347 (1994); Williams, M. E., et al. Science 257:389 (1992); Soong, T. W., et al. Science 260:1133 (1993); Horne, W. A., et al. Proc. Natl. Acad. Sci. USA 90:3787 (1993)) is presented as the strongest example of a general homology of PKD2 to the family of voltage activated Ca 2+ and Na + α1 channel proteins. These channel proteins contain four homologous domains (I-IV), each with six transmembrane spans (S1-S6), which are predicted to form the pore structure (Williams, M. E., et al. (1994), supra; Williams, M. E., et al. (1992), supra; Soong, T. W., et al., supra; Horne, W. A., et al., supra; Hille, B., Ionic channels of excitable membranes (Sinauer Associates, Sunderland, Mass., ed. 2, 1992), pp.250-7)). The membrane spans tm2 through tm6 as well as the intervening intracellular loops of PKD2 have similarity with corresponding segments in the α1 channels (FIG. 4 ). The similarity in the COOH-terminal region includes the putative EF-hand domain ( FIG. 4 ) (Kretsinger, R. H., et al., supra; Babitch, J., supra). This domain in PKD2 scores highly on the EF-hand test ( FIG. 4B ) with identity at all the critical coordination vertices (Kretsinger, R. H., et al., supra; Babitch, J., supra). EF-hand domains are specialized helix-loop-helix motifs that have Ca 2+ binding activity in ˜70% of proteins in which they occur (Nakayama, S. and R. H. Kretsinger Annu. Rev. Biophys. Biomol. Struct. 23:473 (1994)). Unpaired EF-hand sequences have recently been implicated in Ca 2+ -sensitive inactivation of some forms of L-type VACCα1 (de Leon, M., et al. Science 270:1502 (1995)). EF-hand domains that do not coordinate Ca 2+ remain important to protein function (Kretsinger, R. H., et al., supra; Babitch, J., supra; Gulati, A., et al. J. Biol. Chem. 267:25073 (1992)).

Despite the observed homology to PKD1, the predicted structure of the PKD2 protein does not directly suggest a role in cell-cell or matrix-cell signaling similar to that proposed for polycystin (The European Polycystic Kidney Disease Consortium, supra; The International Polycystic Kidney Disease Consortium, supra.; The American PKD1 Consortium, supra; Hughes, J., supra). PKD2 does not have the large NH 2 -terminal extracellular domain and the associated motifs found in polycystin (The European Polycystic Kidney Disease Consortium, supra; The International Polycystic Kidney Disease Consortium, supra; The American PKD1 Consortium, supra; Hughes, J., supra). It is possible that PKD2 functions in a parallel pathway with PKD1. However, given that the clinical diseases produced by mutations in PKD1 and PKD2 exhibit an identical spectrum of organ involvement, differing only in relative rates of progression of cystic changes, hypertension and the development of ESRD, the most likely scenario is that PKD2 associates with itself, with polycystin, and/or with other proteins and ligands as part of a common signal transduction pathway.

PKD2 bears some similarity to the α1 Ca 2+ (and Na + ) channels but has only six membrane spans. If it formed homo- or hetero-multimeric complexes (for example, with itself, with the homologous portion of PKD1 or with another protein), it could function as an ion channel or pore in a manner similar to the K + channels (Hille, B., supra). The observed homologies, the presence of a pair of conserved basic residues (Lys 573, Lys 576) in the fourth transmembrane domain, and the predicted even number of membrane spans, are consistent with such a role (Hille, B., supra). In such a model, PKD1 could act as the regulator of the PKD2 channel activity, perhaps with Ca 2+ as a second messenger in a signal transduction pathway. The discovery of PKD2 raises the possibility that the ADPKD phenotype may in part be the result of a defect in an unknown transport function.

All publications mentioned hereinabove are hereby incorporated by reference in their entirety.

While the foregoing invention has been described in detail for purposes of clarity and understanding, it will be appreciated by one skilled in the art from a reading of the disclosure that various changes in form and detail can be made without departing from the true scope of the invention in the appended claims.

12 866 amino acids amino acid single linear (A) DESCRIPTION peptide NO internal fragment unknown 1Met Val Asn Ser Ser Arg Val Gln Pro Gln1 5 10Gln Pro Gly Asp Ala Lys Arg Pro Pro Ala 15 20Pro Arg Ala Pro Asp Pro Gly Arg Leu Met 25 30Ala Gly Cys Ala Ala Val Gly Ala Ser Leu 35 40Ala Ala Pro Gly Gly Leu Cys Glu Gln Arg 45 50Gly Leu Glu Ile Glu Met Gln Arg Ile Arg 55 60Gln Ala Ala Ala Arg Asp Pro Pro Ala Gly 65 70Ala Ala Ala Ser Pro Ser Pro Pro Leu Ser 75 80Ser Cys Ser Arg Gln Ala Trp Ser Arg Asp 85 90Asn Pro Gly Glu Glu Glu Ala Glu Glu Glu 95 100Glu Glu Glu Val Glu Gly Glu Glu Gly Gly 105 110Met Val Val Glu Met Asp Val Glu Trp Arg 115 120Pro Gly Ser Arg Arg Ser Ala Ala Ser Ser 125 130Ala Val Ser Ser Val Gly Ala Arg Ser Arg 135 140Gly Leu Gly Gly Tyr His Gly Ala Gly His 145 150Pro Ser Gly Arg Arg Arg Arg Arg Glu Asp 155 160Gln Gly Pro Pro Cys Pro Ser Pro Val Gly 165 170Gly Gly Asp Pro Leu His Arg His Leu Pro 175 180Leu Glu Gly Gln Pro Pro Arg Val Ala Trp 185 190Ala Glu Arg Leu Val Arg Gly Leu Arg Gly 195 200Leu Trp Gly Thr Arg Leu Met Glu Glu Ser 205 210Ser Thr Asn Arg Glu Lys Tyr Leu Lys Ser 215 220Val Leu Arg Glu Leu Val Thr Tyr Leu Leu 225 230Phe Leu Ile Val Leu Cys Ile Leu Thr Tyr 235 240Gly Thr Glu Ala Asp Asn Arg Ser Phe Ile 245 250Phe Tyr Glu Asn Leu Leu Leu Gly Val Pro 255 260Arg Ile Arg Gln Leu Arg Val Arg Asn Gly 265 270Ser Cys Ser Ile Pro Gln Asp Leu Arg Asp 275 280Glu Ile Lys Glu Cys Tyr Asp Val Tyr Glu 285 290Thr Ala Ala Gln Val Ala Ser Leu Lys Lys 295 300Asn Val Trp Leu Asp Arg Gly Thr Arg Ala 305 310Thr Phe Ile Asp Phe Ser Val Tyr Asn Ala 315 320Asn Ile Asn Leu Phe Cys Val Val Arg Leu 325 330Leu Val Glu Phe Pro Ala Thr Gly Gly Val 335 340Ile Pro Ser Trp Gln Phe Gln Pro Leu Lys 345 350Leu Ile Arg Tyr Val Thr Thr Phe Asp Phe 355 360Phe Leu Ala Ala Cys Glu Ile Ile Phe Cys 365 370Phe Phe Ile Phe Tyr Tyr Val Val Glu Glu 375 380Ile Leu Glu Xaa Ile Arg Ile His Lys Leu 385 390His Tyr Phe Arg Xaa Ser Phe Trp Asn Cys 395 400Leu Asp Val Val Ile Val Val Leu Ser Val 405 410Val Ala Ile Gly Ile Asn Ile Tyr Arg Thr 415 420Ser Asn Val Glu Val Xaa Leu Leu Gln Phe 425 430Leu Xaa Glu Asp Gln Asn Thr Phe Pro Asn 435 440Phe Glu His Leu Ala Tyr Trp Gln Ile Gln 445 450Phe Asn Asn Ile Ala Ala Val Thr Val Phe 455 460Phe Val Trp Ile Lys Leu Phe Lys Phe Ile 465 470Asn Phe Asn Arg Thr Met Ser Gln Leu Ser 475 480Thr Thr Met Ser Arg Cys Ala Lys Asp Leu 485 490Phe Gly Phe Ala Ile Met Phe Phe Ile Ile 495 500Phe Leu Ala Tyr Ala Gln Leu Ala Tyr Leu 505 510Val Phe Gly Thr Gln Val Asp Asp Phe Ser 515 520Thr Phe Gln Glu Cys Ile Phe Thr Gln Phe 525 530Arg Ile Ile Leu Gly Asp Ile Asn Phe Ala 535 540Glu Ile Glu Glu Ala Asn Xaa Arg Val Leu 545 550Gly Pro Ile Tyr Phe Thr Thr Phe Val Phe 555 560Phe Met Phe Phe Ile Leu Leu Asn Met Phe 565 570Leu Ala Ile Ile Asn Asp Thr Tyr Ser Glu 575 580Val Lys Ser Asp Leu Xaa Xaa Xaa Ala Gln 585 590Gln Lys Ala Glu Met Glu Leu Ser Asp Leu 595 600Ile Arg Lys Gly Tyr His Lys Ala Leu Val 605 610Lys Leu Lys Leu Lys Lys Asn Thr Val Asp 615 620Asp Ile Ser Glu Ser Leu Arg Gln Gly Gly 625 630Gly Lys Leu Asn Phe Asp Glu Leu Arg Gln 635 640Asp Leu Lys Gly Lys Gly His Thr Asp Ala 645 650Glu Ile Glu Ala Ile Phe Thr Lys Tyr Asp 655 660Gln Asp Gly Asp Gln Glu Leu Thr Glu His 665 670Glu His Gln Gln Met Arg Asp Asp Leu Glu 675 680Lys Glu Arg Glu Asp Leu Asp Leu Asp His 685 690Ser Ser Leu Pro Arg Pro Met Ser Ser Arg 695 700Ser Phe Pro Arg Ser Leu Asp Asp Ser Glu 705 710Glu Asp Asp Asp Glu Asp Ser Gly His Ser 715 720Ser Arg Arg Arg Gly Ser Ile Ser Ser Gly 725 730Val Ser Tyr Glu Glu Phe Gln Val Leu Val 735 740Arg Arg Val Asp Arg Met Glu His Ser Ile 745 750Gly Ser Ile Val Ser Lys Ile Asp Ala Val 755 760Ile Val Lys Leu Glu Ile Met Glu Arg Ala 765 770Lys Leu Lys Arg Arg Glu Val Leu Gly Arg 775 780Leu Leu Asp Gly Val Ala Glu Asp Glu Arg 785 790Leu Gly Arg Asp Ser Glu Ile His Arg Glu 795 800Gln Met Glu Arg Leu Val Arg Glu Glu Leu 805 810Glu Arg Trp Glu Ser Asp Asp Ala Ala Ser 815 820Gln Ile Ser His Gly Leu Gly Thr Pro Val 825 830Gly Leu Asn Gly Gln Pro Arg Pro Arg Ser 835 840Ser Arg Pro Ser Ser Ser Gln Ser Xaa Thr 845 850Glu Gly Met Glu Gly Ala Gly Gly Asn Gly 855 860Ser Ser Asn Val His Val 865 523 amino acids amino acid single linear (A) DESCRIPTION peptide NO internal fragment unknown 2Phe Leu Ala Lys Glu Glu Ala Arg Lys Val1 5 10Lys Arg Leu His Gly Met Leu Arg Ser Leu 15 20Leu Val Tyr Met Leu Phe Leu Leu Val Thr 25 30Leu Leu Ala Ser Tyr Gly Asp Ala Ser Cys 35 40His Gly His Ala Tyr Xaa Arg Leu Gln Ser 45 50Xaa Xaa Xaa Xaa Xaa Ala Ile Lys Gln Glu 55 60Leu His Ser Arg Ala Phe Leu Ala Ile Thr 65 70Arg Ser Glu Glu Leu Trp Pro Trp Met Ala 75 80His Val Leu Leu Pro Tyr Val His Xaa Xaa 85 90Xaa Xaa Xaa Gly Asn Gln Ser Ser Pro Glu 95 100Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 105 110Xaa Leu Gly Pro Pro Arg Leu Arg Gln Val 115 120Arg Leu Gln Glu Ala Leu Tyr Pro Asp Pro 125 130Pro Gly Pro Arg Val His Thr Cys Ser Ala 135 140Ala Gly Gly Phe Ser Thr Ser Asp Tyr Asp 145 150Val Gly Trp Glu Ser Pro His Asn Gly Ser 155 160Gly Thr Trp Ala Thr Xaa Xaa Ser Ala Pro 165 170Asp Leu Leu Gly Ala Trp Ser Trp Gly Ser 175 180Cys Ala Val Tyr Asp Ser Gly Gly Tyr Val 185 190Gln Glu Leu Gly Leu Ser Leu Glu Glu Ser 195 200Arg Asp Arg Leu Arg Phe Leu Gln Leu His 205 210Asn Trp Leu Asp Asn Arg Ser Arg Ala Val 215 220Phe Leu Glu Leu Thr Arg Tyr Ser Pro Ala 225 230Val Gly Leu His Ala Ala Val Thr Leu Arg 235 240Leu Glu Phe Pro Ala Ala Gly Arg Ala Leu 245 250Ala Ala Leu Ser Val Arg Pro Phe Ala Leu 255 260Arg Arg Leu Ser Ala Gly Leu Ser Leu Pro 265 270Leu Leu Thr Ser Val Cys Leu Leu Leu Phe 275 280Ala Val His Phe Ala Val Ala Glu Ala Arg 285 290Thr Trp Xaa His Arg Glu Gly Arg Trp Arg 295 300Val Leu Arg Leu Gly Ala Trp Ala Arg Trp 305 310Leu Leu Val Ala Leu Thr Ala Ala Thr Ala 315 320Leu Val Arg Leu Ala Gln Leu Gly Ala Ala 325 330Asp Arg Gln Xaa Xaa Trp Thr Arg Phe Val 335 340Arg Gly Arg Pro Arg Arg Phe Thr Ser Phe 345 350Asp Gln Val Ala Gln Leu Ser Ser Ala Ala 355 360Arg Gly Leu Ala Ala Ser Leu Leu Phe Leu 365 370Leu Leu Val Lys Ala Ala Gln Gln Leu Arg 375 380Phe Val Arg Gln Trp Ser Val Phe Gly Lys 385 390Thr Leu Cys Arg Ala Leu Pro Glu Leu Leu 395 400Gly Val Thr Leu Gly Leu Val Val Leu Gly 405 410Val Ala Tyr Ala Gln Leu Ala Ile Leu Leu 415 420Val Ser Ser Cys Val Asp Ser Leu Trp Ser 425 430Val Ala Gln Ala Leu Leu Xaa Xaa Xaa Xaa 435 440Val Leu Cys Pro Gly Thr Gly Leu Ser Thr 445 450Leu Cys Pro Ala Glu Ser Trp His Leu Ser 455 460Pro Leu Leu Cys Val Gly Leu Trp Ala Leu 465 470Arg Leu Trp Gly Ala Leu Arg Leu Gly Ala 475 480Val Ile Leu Arg Trp Arg Tyr His Ala Leu 485 490Arg Gly Glu Leu Tyr Arg Pro Ala Trp Glu 495 500Pro Gln Asp Tyr Glu Met Val Glu Leu Phe 505 510Xaa Xaa Xaa Xaa Xaa Xaa Xaa Leu Arg Arg 515 520Leu Arg Leu 399 amino acids amino acid single linear (A) DESCRIPTION peptide NO internal fragment unknown 3Glu Asn Arg Lys Met Arg Asp Glu Gln Leu1 5 10Phe Ile Thr Ile Arg Asp Met Leu Cys Phe 15 20Phe Ala Ser Leu Tyr Ile Met Val Met Leu 25 30Thr Tyr Tyr Cys Lys Asp Arg His Gly Tyr 35 40Trp Tyr Gln Leu Glu Met Ser Thr Ile Leu 45 50Asn Ile Asn Gln Lys Asn Tyr Gly Asp Asn 55 60Thr Xaa Phe Met Ser Ile Gln His Ala Asp 65 70Asp Phe Trp Asp Trp Ala Arg Glu Ser Leu 75 80Ala Thr Ala Leu Leu Ala Ser Trp Tyr Asp 85 90Gly Asn Pro Ala Tyr Gly Met Arg Ala Tyr 95 100Met Asn Asp Lys Val Ser Arg Ser Met Gly 105 110Ile Gly Thr Ile Arg Gln Val Arg Thr Lys 115 120Lys Ser Glu Ile Ile Thr Leu Phe Asn Lys 125 130Leu Asp Ser Glu Arg Trp Ile Asp Asp His 135 140Thr Arg Ala Val Ile Ile Glu Phe Ser Ala 145 150Tyr Asn Ala Gln Ile Asn Tyr Phe Ser Val 155 160Val Gln Leu Leu Val Glu Ile Pro Lys Ser 165 170Gly Ile Tyr Leu Pro Asn Ser Trp Val Glu 175 180Ser Val Arg Leu Ile Lys Ser Glu Gly Ser 185 190Asp Gly Thr Val Val Lys Tyr Tyr Glu Met 195 200Leu Tyr Ile Phe Phe Ser Val Leu Ile Phe 205 210Val Lys Glu Ile Val Trp Asn Phe Met Asp 215 220Leu Ile Val Gly Ala Leu Ala Val Ala Ser 225 230Val Leu Ala Tyr Thr Ile Arg Gln Arg Thr 235 240Thr Asn Arg Ala Met Glu Asp Phe Asn Ala 245 250Asn Asn Gly Asn Ser Tyr Ile Asn Leu Thr 255 260Glu Gln Arg Asn Trp Glu Ile Val Phe Ser 265 270Tyr Cys Leu Ala Gly Ala Val Phe Phe Thr 275 280Ser Cys Lys Met Ile Arg Ile Leu Arg Phe 285 290Asn Arg Arg Ile Gly Val Leu Ala Ala Thr 295 300Leu Asp Asn Ala Leu Gly Ala Ile Val Ser 305 310Phe Gly Ile Ala Phe Leu Phe Phe Ser Met 315 320Thr Phe Asn Ser Val Leu Tyr Ala Val Leu 325 330Gly Asn Lys Met Gly Gly Tyr Arg Ser Leu 335 340Met Ala Thr Phe Gln Thr Ala Leu Ala Gly 345 350Met Leu Gly Lys Leu Asp Val Thr Ser Ile 355 360Gln Pro Xaa Xaa Xaa Xaa Xaa Ile Ser Gln 365 370Phe Ala Phe Val Val Ile Met Leu Tyr Met 375 380Ile Glu Phe Glu Glu Ile Arg Asn Asp Ser 385 390Glu Lys Gln Thr Asn Asp Tyr Glu Ile 395 363 amino acids amino acid single linear (A) DESCRIPTION peptide NO internal fragment unknown 4Phe Thr Met Val Phe Ser Leu Glu Cys Val1 5 10Leu Lys Val Ile Ala Phe Gly Phe Leu Asn 15 20Tyr Phe Arg Xaa Asp Thr Trp Asn Ile Phe 25 30Asp Phe Ile Thr Val Ile Gly Ser Ile Thr 35 40Glu Ile Ile Leu Thr Asp Ser Lys Leu Val 45 50Asn Thr Ser Gly Xaa Phe Xaa Xaa Xaa Xaa 55 60Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 65 70Xaa Asn Met Ser Phe Leu Lys Xaa Xaa Xaa 75 80Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Leu Phe 85 90Arg Ala Ala Arg Leu Ile Lys Leu Leu Arg 95 100Gln Gly Tyr Thr Ile Arg Ile Leu Leu Trp 105 110Thr Phe Val Gln Ser Phe Lys Ala Leu Pro 115 120Tyr Val Cys Leu Leu Ile Ala Met Leu Phe 125 130Phe Ile Tyr Ala Ile Ile Gly Met Gln Val 135 140Phe Gly Asn Asn Phe Arg Ser Phe Phe Gly 145 150Ser Leu Met Leu Leu Phe Arg Ser Ala Thr 155 160Gly Glu Xaa Ala Trp Gln Glu Ile Glu Arg 165 170Cys Gly Xaa Thr Asp Leu Ala Tyr Val Tyr 175 180Phe Val Ser Phe Ile Phe Phe Cys Ser Phe 185 190Leu Met Leu Asn Leu Phe Val Ala Val Ile 195 200Met Asp Asn Phe Glu Tyr Leu Thr Arg Asp 205 210Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 215 220Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 225 230Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 235 240Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 245 250Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 255 260Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ile Leu Gly 265 270Pro His His Leu Asp Xaa Glu Phe Val Arg 275 280Val Trp Ala Glu Tyr Asp Arg Ala Ala Cys 285 290Gly Arg Ile His Tyr Thr Glu Met Tyr Glu 295 300Met Glu Arg Arg Arg Ser Lys Glu Arg Lys 305 310His Leu Leu Ser Pro Asp Val Ser Arg Cys 315 320Asn Ser Glu Glu Arg Gly Thr Gln Ala Asp 325 330Trp Glu Ser Pro Glu Arg Arg Gln Ser Arg 335 340Ser Pro Ser Glu Gly Arg Ser Gln Thr Pro 345 350Asn Arg Gln Gly Thr Gly Ser Leu Ser Glu 355 360Ser Ser Ile 28 amino acids amino acid single linear (A) DESCRIPTION peptide NO unknown 5Glu Ile Ala Ile Phe Thr Lys Tyr Asp Gln1 5 10Asp Gly Asp Gln Glu Leu Thr Glu His Glu 15 20His Gln Gln Met Arg Asp Asp Leu 25 5057 nucleic acids nucleic acid single linear (A) DESCRIPTION genomic DNA NO unknown 6GGCTCCTGAG GCGCACAGCG CCGAGCGCGG CGCCGCGCAC CCGCGCGCCG 50GACGCCAGTG ACCGCGATGG TGAACTCCAG TCGCGTGCAG CCTCAGCAGC 100CCGGGGACGC CAAGCGGCCG CCCGCGCCCC GCGCGCCGGA CCCGGGCCGG 150CTGATGGCTG GCTGCGCGGC CGTGGGCGCC AGCCTCGCCG CCCCGGGCGG 200CCTCTGCGAG CAGCGGGGCC TGGAGATCGA GATGCAGCGC ATCCGGCAGG 250CGGCCGCGCG GGACCCCCCG GCCGGAGCCG CGGCCTCCCC TTCTCCTCCG 300CTCTCGTCGT GCTCCCGGCA GGCGTGGAGC CGCGATAACC CCGGCTTCGA 350GGCCGAGGAG GAGGAGGAGG AGGTGGAAGG GGAAGAAGGC GGAATGGTGG 400TGGAGATGGA CGTAGAGTGG CGCCCGGGCA GCCGGAGGTC GGCCGCCTCC 450TCGGCCGTGA GCTCCGTGGG CGCGCGGAGC CGGGGGCTTG GGGGCTACCA 500CGGCGCGGGC CACCCGAGCG GGAGGCGGCG CCGGCGAGAG GACCAGGGCC 550CGCCGTGCCC CAGCCCAGTC GGCGGCGGGG ACCCGCTGCA TCGCCACCTC 600CCCCTGGAAG GGCAGCCGCC CCGAGTGGCC TGGGCGGAGA GGCTGGTTCG 650CGGGCTGCGA GGTCTCTGGG GAACAAGACT CATGGAGGAA AGCAGCACTA 700ACCGAGAGAA ATACCTTAAA AGTGTTTTAC GGGAACTGGT CACATACCTC 750CTTTTTCTCA TAGTCTTGTG CATCTTGACC TACGGCATGA TGAGCTCCAA 800TGTGTACTAC TACACCCGGA TGATGTCACA GCTCTTCCTA GACACCCCCG 850TGTCCAAAAC GGAGAAAACT AACTTTAAAA CTCTGTCTTC CATGGAAGAC 900TTCTGGAAGT TCACAGAAGG CTCCTTATTG GATGGGCTGT ACTGGAAGAT 950GCAGCCCAGC AACCAGACTG AAGCTGACAA CCGAAGTTTC ATCTTCTATG 1000AGAACCTGCT GTTAGGGGTT CCACGAATAC GGCAACTCCG AGTCAGAAAT 1050GGATCCTGCT CTATCCCCCA GGACTTGAGA GATGAAATTA AAGAGTGCTA 1100TGATGTCTAC TCTGTCAGTA GTGAAGATAG GGCTCCCTTT GGGCCCCGAA 1150ATGGAACCGC TTGGATCTAC ACAAGTGAAA AAGACTTGAA TGGTAGTAGC 1200CACTGGGGAA TCATTGCAAC TTATAGTGGA GCTGGCTATT ATCTGGATTT 1250GTCAAGAACA AGAGAGGAAA CAGCTGCACA AGTTGCTAGC CTCAAGAAAA 1300ATGTCTGGCT GGACCGAGGA ACCAGGGCAA CTTTTATTGA CTTCTCAGTG 1350TACAACGCCA ACATTAACCT GTTCTGTGTG GTCAGGTTAT TGGTTGAATT 1400CCCAGCAACA GGTGGTGTGA TTCCATCTTG GCAATTTCAG CCTTTAAAGC 1450TGATCCGATA TGTCACAACT TTTGATTTCT TCCTGGCAGC CTGTGAGATT 1500ATCTTTTGTT TCTTTATCTT TTACTATGTG GTGGAAGAGA TATTGGAAAT 1550TCGCATTCAC AAACTACACT ATTTCAGGAG TTTCTGGAAT TGTCTGGATG 1600TTGTGATCGT TGTGCTGTCA GTGGTAGCTA TAGGAATTAA CATATACAGA 1650ACATCAAATG TGGAGGTGCT ACTACAGTTT CTGGAAGATC AAAATACTTT 1700CCCCAACTTT GAGCATCTGG CATATTGGCA GATACAGTTC AACAATATAG 1750CTGCTGTCAC AGTATTTTTT GTCTGGATTA AGCTCTTCAA ATTCATCAAT 1800TTTAACAGGA CCATGAGCCA GCTCTCGACA ACCATGTCTC GATGTGCCAA 1850AGACCTGTTT GGCTTTGCTA TTATGTTCTT CATTATTTTC CTAGCGTATG 1900CTCAGTTGGC ATACCTTGTC TTTGGCACTC AGGTCGATGA CTTCAGTACT 1950TTCCAAGAGT GTATCTTCAC TCAATTCCGT ATCATTTTGG GCGATATCAA 2000CTTTGCAGAG ATTGAGGAAG CTAATCGAGT TTTGGGACCA ATTTATTTCA 2050CTACATTTGT GTTCTTTATG TTCTTCATTC TTTTGAATAT GTTTTTGGCT 2100ATCATCAATG ATACTTACTC TGAAGTGAAA TCTGACTTGG CACAGCAGAA 2150AGCTGAAATG GAACTCTCAG ATCTTATCAG AAAGGGCTAC CATAAAGCTT 2200TGGTCAAACT AAAACTGAAA AAAAATACCG TGGATGACAT TTCAGAGAGT 2250CTGCGGCAAG GAGGAGGCAA GTTAAACTTT GACGAACTTC GACAAGATCT 2300CAAAGGGAAG GGCCATACTG ATGCAGAGAT TGAGGCAATA TTCACAAAGT 2350ACGACCAAGA TGGAGACCAA GAACTGACCG AACATGAACA TCAGCAGATG 2400AGAGACGACT TGGAGAAAGA GAGGGAGGAC CTGGATTTGG ATCACAGTTC 2450TTTACCACGT CCCATGAGCA GCCGAAGTTT CCCTCGAAGC CTGGATGACT 2500CTGAGGAGGA TGACGATGAA GATAGCGGAC ATAGCTCCAG AAGGAGGGGA 2550AGCATTTCTA GTGGCGTTTC TTACGAAGAG TTTCAAGTCC TGGTGAGACG 2600AGTGGACCGG ATGGAGCATT CCATCGGCAG CATAGTGTCC AAGATTGACG 2650CCGTGATCGT GAAGCTAGAG ATTATGGAGC GAGCCAAACT GAAGAGGAGG 2700GAGGTGCTGG GAAGGCTGTT GGATGGGGTG GCCGAGGATG AAAGGCTGGG 2750TCGTGACAGT GAAATCCATA GGGAACAGAT GGAACGGCTA GTACGTGAAG 2800AGTTGGAACG CTGGGAATCC GATGATGCAG CTTCCCAGAT CAGTCATGGT 2850TTAGGCACGC CAGTGGGACT AAATGGTCAA CCTCGCCCCA GAAGCTCCCG 2900CCCATCTTCC TCCCAATCTA CAGAAGGCAT GGAAGGTGCA GGTGGAAATG 2950GGAGTTCTAA TGTCCACGTA TGATATGTGT GTTTCAGTAT GTGTGTTTCT 3000AATAAGTGAG GAAGTGGCTG TCCTGAATTG CTGTAACAAG CACACTATTT 3050ATATGCCCTG ACCACCATAG GATGCTAGTC TTTGTGACCG ATTGCTAATC 3100TTCTGCACTT TAATTTATTT TATATAAACT TTACCCATGG TTCAAAGATT 3150TTTTTTTCTT TTTCTCATAT AAGAAATCTA GGTGTAAATA TTGAGTACAG 3200AAAAAAAATC TTCATGATGT GTATTGAGCG GTACGCCCAG TTGCCACCAT 3250GACTGAGTCT TCTCAGTTGA CAATGAAGTA GCCTTTTAAA GCTAGAAAAC 3300TGTCAAAGGG CTTCTGAGTT TCATTTCCAG TCACAAAAAT CAGTATTGTT 3350ATTTTTTTCC AAGAGTGTGA AGGAAAATGG GGCAATTCCT TTCCACTCTG 3400GCATAGTTCA TGAGCTTAAT ACATAGCTTT CTTTTAAGAA AGGAGCCTTT 3450TTTTTCAACT AGCTTCCTGG GGTAAACTTT TCTAAAAGAT AAAATGGGAA 3500GGAACTCCAA ACTATGATAG AATCTGTGTG AATGGTTAAG ATGAATGTTA 3550AATACTATGC TTTTTTGTAA GTTGATCGTA TCTGATGTCT GTGGGACTAA 3600CTGTATCACT TAATTTTTAC CTTATTTTGG CTCTAATTTG AATAAGCTGA 3650GTAAAACCAC CAAAGATCAG TTATAGGATA AAATGGCATC TCTAACCATA 3700ACACAGGAGA ATTGGAAGGA GCCCTAAGTT GTCACTCAGT TTAATTTCTT 3750TTAATGGTTA GTTTAGCCTA AAGATTTATC TGCATATTCT TTTTCCCATG 3800TGGCTCTACT CATTTGCAAC TGAATTTAAT GTTATAACTC ATCTAGTGAG 3850ACCAACTTAC TAAATTTTTA GTATGCACTG AAAGTTTTTA TCCAACAATT 3900ATGTTCATTT TAAGCAAAAT TTTAAGAAAG TTTTGAAATT CATAAAGCAT 3950TTGGTTTTAA ACTATTTTAA GAATATAGTA CTCGGTCAGG TATGNNNCAC 4000GCCTGTAATC CCAGCACTTT GGGAGGCCGA AACAGGCGAA TCACTTGAGC 4050CCAGGAGTTC AAGACCAACA TGGGCAATGT GGCGAAACTC CATCTCTACA 4100AAAAATGCAA AAATAAAAAA TATAGTACTC AAGTATTCTT GATCCTGTGT 4150TTCAAAACTA GAATTTGTAA TGCAAATGGA GCTCAGTCTA ATAAAAAAGA 4200GGTTTTGGTA TTAAAAGTTC ATACATTAGA CAGTATCAGC CAAAATTTGA 4250GTTAGCAACA CTGTTTTCTT TACGAGAGGG TCTCACCCAA ATTTATGGGG 4300AGAAATCTAT TTCTCAAAAA AAAAAAATCT TCTTTTACAG AAATGTTGAG 4350TAAGGTGACA TTTTGAGCGC TAATAAGCAA AAGAGCATGC AGTGCTGTTG 4400AATAACCCTC ACTTGGAGAA CCAAGAGAAT CCTGTCGTTT AATGCTATAT 4450TTTAATTTCA CAAGTTGTTC ATTTAACTGG TAGAATGTCA GTCCAATCTC 4500CAATGAGAAC ATGAGCAAAT AGACCTTTCC AGGTTGAAAG TGAAACATAC 4550TGGGTTTCTG TAAGTTTTTC CTCATGGCTT CATCTCTATC TTTACTTTCT 4600CTTGAATATG CTACACAAAG TTCTTTATTA CTACATACTA AAGTTTGCAT 4650TCCAGGGATA TTGACTGTAC ATATTTATGT ATATGTACCA TGTTGTTACA 4700TGTAAACAAA CTTCAATTTG AAGTGCAGCT ATTATGTGGT ATCCATGTGT 4750ATCGACCATG TGCCATATAT CAATTATGGT CACTAGAAAG TCTCTTTATG 4800ATACTTTTTA TTGTACTGTT TTTCATTTCA CTTGCAAAAT TTTGCAGAAT 4850TCCTCCTTTC TACCCATAAA TTACATATAA TTTTTCTTCT TTAGTCATGG 4900AGAACNCCCC CCCATCATCT CANCCCTATT ANCTTTCCCA TGTGTACTGG 4950TATTATTAAA AAGACATTTA CATACGCAAG TTTTTCACTG ACAANCAAGA 5000ATGTTATTAA TGTGTAATAC TGAGCACNTT TACTTCTTAA TAAAAACTTG 5050ATATANT 5057 968 amino acids amino acid single linear (A) DESCRIPTION peptide NO unknown 7Met Val Asn Ser Ser Arg Val Gln Pro Gln Gln Pro Gly Asp Ala Lys1 5 10 15Arg Pro Pro Ala Pro Arg Ala Pro Asp Pro Gly Arg Leu Met Ala Gly 20 25 30Cys Ala Ala Val Gly Ala Ser Leu Ala Ala Pro Gly Gly Leu Cys Glu 35 40 45Gln Arg Gly Leu Glu Ile Glu Met Gln Arg Ile Arg Gln Ala Ala Ala 50 55 60Arg Asp Pro Pro Ala Gly Ala Ala Ala Ser Pro Ser Pro Pro Leu Ser65 70 75 80Ser Cys Ser Arg Gln Ala Trp Ser Arg Asp Asn Pro Gly Phe Glu Ala 85 90 95Glu Glu Glu Glu Glu Glu Val Glu Gly Glu Glu Gly Gly Met Val Val 100 105 110Glu Met Asp Val Glu Trp Arg Pro Gly Ser Arg Arg Ser Ala Ala Ser 115 120 125Ser Ala Val Ser Ser Val Gly Ala Arg Ser Arg Gly Leu Gly Gly Tyr 130 135 140His Gly Ala Gly His Pro Ser Gly Arg Arg Arg Arg Arg Glu Asp Gln145 150 155 160Gly Pro Pro Cys Pro Ser Pro Val Gly Gly Gly Asp Pro Leu His Arg 165 170 175His Leu Pro Leu Glu Gly Gln Pro Pro Arg Val Ala Trp Ala Glu Arg 180 185 190Leu Val Arg Gly Leu Arg Gly Leu Trp Gly Thr Arg Leu Met Glu Glu 195 200 205Ser Ser Thr Asn Arg Glu Lys Tyr Leu Lys Ser Val Leu Arg Glu Leu 210 215 220Val Thr Tyr Leu Leu Phe Leu Ile Val Leu Cys Ile Leu Thr Tyr Gly225 230 235 240Met Met Ser Ser Asn Val Tyr Tyr Tyr Thr Arg Met Met Ser Gln Leu 245 250 255Phe Leu Asp Thr Pro Val Ser Lys Thr Glu Lys Thr Asn Phe Lys Thr 260 265 270Leu Ser Ser Met Glu Asp Phe Trp Lys Phe Thr Glu Gly Ser Leu Leu 275 280 285Asp Gly Leu Tyr Trp Lys Met Gln Pro Ser Asn Gln Thr Glu Ala Asp 290 295 300Asn Arg Ser Phe Ile Phe Tyr Glu Asn Leu Leu Leu Gly Val Pro Arg305 310 315 320Ile Arg Gln Leu Arg Val Arg Asn Gly Ser Cys Ser Ile Pro Gln Asp 325 330 335Leu Arg Asp Glu Ile Lys Glu Cys Tyr Asp Val Tyr Ser Val Ser Ser 340 345 350Glu Asp Arg Ala Pro Phe Gly Pro Arg Asn Gly Thr Ala Trp Ile Tyr 355 360 365Thr Ser Glu Lys Asp Leu Asn Gly Ser Ser His Trp Gly Ile Ile Ala 370 375 380Thr Tyr Ser Gly Ala Gly Tyr Tyr Leu Asp Leu Ser Arg Thr Arg Glu385 390 395 400Glu Thr Ala Ala Gln Val Ala Ser Leu Lys Lys Asn Val Trp Leu Asp 405 410 415Arg Gly Thr Arg Ala Thr Phe Ile Asp Phe Ser Val Tyr Asn Ala Asn 420 425 430Ile Asn Leu Phe Cys Val Val Arg Leu Leu Val Glu Phe Pro Ala Thr 435 440 445Gly Gly Val Ile Pro Ser Trp Gln Phe Gln Pro Leu Lys Leu Ile Arg 450 455 460Tyr Val Thr Thr Phe Asp Phe Phe Leu Ala Ala Cys Glu Ile Ile Phe465 470 475 480Cys Phe Phe Ile Phe Tyr Tyr Val Val Glu Glu Ile Leu Glu Ile Arg 485 490 495Ile His Lys Leu His Tyr Phe Arg Ser Phe Trp Asn Cys Leu Asp Val 500 505 510Val Ile Val Val Leu Ser Val Val Ala Ile Gly Ile Asn Ile Tyr Arg 515 520 525Thr Ser Asn Val Glu Val Leu Leu Gln Phe Leu Glu Asp Gln Asn Thr 530 535 540Phe Pro Asn Phe Glu His Leu Ala Tyr Trp Gln Ile Gln Phe Asn Asn545 550 555 560Ile Ala Ala Val Thr Val Phe Phe Val Trp Ile Lys Leu Phe Lys Phe 565 570 575Ile Asn Phe Asn Arg Thr Met Ser Gln Leu Ser Thr Thr Met Ser Arg 580 585 590Cys Ala Lys Asp Leu Phe Gly Phe Ala Ile Met Phe Phe Ile Ile Phe 595 600 605Leu Ala Tyr Ala Gln Leu Ala Tyr Leu Val Phe Gly Thr Gln Val Asp 610 615 620Asp Phe Ser Thr Phe Gln Glu Cys Ile Phe Thr Gln Phe Arg Ile Ile625 630 635 640Leu Gly Asp Ile Asn Phe Ala Glu Ile Glu Glu Ala Asn Arg Val Leu 645 650 655Gly Pro Ile Tyr Phe Thr Thr Phe Val Phe Phe Met Phe Phe Ile Leu 660 665 670Leu Asn Met Phe Leu Ala Ile Ile Asn Asp Thr Tyr Ser Glu Val Lys 675 680 685Ser Asp Leu Ala Gln Gln Lys Ala Glu Met Glu Leu Ser Asp Leu Ile 690 695 700Arg Lys Gly Tyr His Lys Ala Leu Val Lys Leu Lys Leu Lys Lys Asn705 710 715 720Thr Val Asp Asp Ile Ser Glu Ser Leu Arg Gln Gly Gly Gly Lys Leu 725 730 735Asn Phe Asp Glu Leu Arg Gln Asp Leu Lys Gly Lys Gly His Thr Asp 740 745 750Ala Glu Ile Glu Ala Ile Phe Thr Lys Tyr Asp Gln Asp Gly Asp Gln 755 760 765Glu Leu Thr Glu His Glu His Gln Gln Met Arg Asp Asp Leu Glu Lys 770 775 780Glu Arg Glu Asp Leu Asp Leu Asp His Ser Ser Leu Pro Arg Pro Met785 790 795 800Ser Ser Arg Ser Phe Pro Arg Ser Leu Asp Asp Ser Glu Glu Asp Asp 805 810 815Asp Glu Asp Ser Gly His Ser Ser Arg Arg Arg Gly Ser Ile Ser Ser 820 825 830Gly Val Ser Tyr Glu Glu Phe Gln Val Leu Val Arg Arg Val Asp Arg 835 840 845Met Glu His Ser Ile Gly Ser Ile Val Ser Lys Ile Asp Ala Val Ile 850 855 860Val Lys Leu Glu Ile Met Glu Arg Ala Lys Leu Lys Arg Arg Glu Val865 870 875 880Leu Gly Arg Leu Leu Asp Gly Val Ala Glu Asp Glu Arg Leu Gly Arg 885 890 895Asp Ser Glu Ile His Arg Glu Gln Met Glu Arg Leu Val Arg Glu Glu 900 905 910Leu Glu Arg Trp Glu Ser Asp Asp Ala Ala Ser Gln Ile Ser His Gly 915 920 925Leu Gly Thr Pro Val Gly Leu Asn Gly Gln Pro Arg Pro Arg Ser Ser 930 935 940Arg Pro Ser Ser Ser Gln Ser Thr Glu Gly Met Glu Gly Ala Gly Gly945 950 955 960Asn Gly Ser Ser Asn Val His Val 965 19 nucleic acids nucleic acid single linear (A) DESCRIPTION genomic DNA NO unknown 8GGGCTACCAT AAAGCTTTG 19 20 nucleic acids nucleic acid single linear (A) DESCRIPTION genomic DNA NO unknown 9GTTCATGTTC GATCAGTTCT 20 23 nucleic acids nucleic acid single linear (A) DESCRIPTION genomic DNA NO unknown 10GGGCTAGAAA TACTCTTATC ACC 23 20 nucleic acids nucleic acid single linear (A) DESCRIPTION genomic DNA NO unknown 11GCCTCAAGTG TTCCACTGAT 20 22 nucleic acids nucleic acid single linear (A) DESCRIPTION genomic DNA NO unknown 12AGGTTTTTCT GGGTAACCCT AG 22

Claims

1. A method of detecting the presence of a mutation in the sequence of the PKD2 gene (SEQ ID NO:6) comprising the steps of:a) obtaining a polynucleotide sample from a subject; b) comparing the polynucleotide sample to a reference wild-type PKD2 sequence (SEQ ID NO:6); and c) determining the differences between the polynucleotide sample and the reference wild-type PKD2 sequence wherein the differences are mutations which comprise one or more deletion, insertion, point or rearrangement mutations.

2. The method of claim 1 wherein the subject is an embryo, fetus, newborn, infant or adult.

3. The method of claim 1, wherein the polynucleotide is DNA or RNA.