SPECIAL ARTICLE www.jasn.org

2008 Homer W. Smith Award: Insights into the Pathogenesis of Polycystic Kidney Disease from Discovery

Peter C. Harris

Division of Nephrology and Hypertension, Mayo Clinic, Rochester, Minnesota

ABSTRACT Polycystic kidney diseases (PKD) are a group of inherited disorders characterized autosomal dominant PKD (ADPKD; by morbidity-associated development of renal cysts. Three forms of PKD are adult PKD as it was known then), when described here: The common, late onset, autosomal dominant PKD (ADPKD); the Steve Reeders visited Children’s Hospital mainly infantile, autosomal recessive PKD (ARPKD); and the lethal, syndromic, and presented his work from Oxford Meckel syndrome that also includes central nervous system and digital defects. (later published in Nature3) of linkage of Positional cloning approaches based on genetic linkage have identified the disease an ADPKD gene to 16, just in these disorders. Completion of the Project, cases with 5 cM from the ␣-globin locus. So started atypical mutation, and animal models have greatly aided gene identification, and a project, initially to identify additional characterization of the disease genes has allowed establishment of molecular markers (rather unsuccessfully) to local- diagnostics. Genetic and allelic heterogeneity, plus genetic modification, underlie ize this gene precisely—which became the significant phenotypic variability in each disorder. Positional cloning identified PKD1 once it was clear that ADPKD was novel disease-associated families: The polycystins (ADPKD); fibrocystins a genetically heterogeneous disease. In (ARPKD); and meckelin. A common feature of pathogenesis in each disorder seems 1987, I moved as a postdoctoral fellow to to be the primary cilia, implicating detection of fluid flow and the developmental Oxford, whereupon Reeders rapidly left process of planar cell polarity. Identifying the primary defect has contributed to for Yale, leaving me an unexpected de- our understanding of defective cellular processes and highlights potential thera- gree of freedom. Doug Higgs (a molecu- peutic targets. A number of agents are now in Phase 3 trials, and many others show lar hematologist interested in genomics) promise preclinically, providing hope of effective treatments for ADPKD in the took me under his wing, and I found a foreseeable future. home in the Institute of Molecular Med- icine of David Weatherall (now the J Am Soc Nephrol 20: 1188–1198, 2009. doi: 10.1681/ASN.2009010014 Weatherall Institute of Molecular Medi- cine). Higgs taught me the importance of observation, of asking important ques- The twists and turns of a research career back then) was just starting to show tions, and of building a patient base to and the ultimate direction it takes often promise to identify the primary defect in support the project. Despite Weatherall’s resemble happenstance rather than a common monogenic disorders. As well attempts to redirect my career away from carefully choreographed life plan. In my as temporal considerations, spatial fac- PKD, he helped establish collaborations case, chance factors resulted in a quarter- tors were a strong influence. My first with Peter Ratcliffe and, in turn, with century musing about renal cystic dis- postdoctoral position with Sam Latt at Chris Winearls, the clinical director of eases. My undergraduate training in ge- Children’s Hospital, Boston, was just the Oxford Kidney Unit. This was the netics at the University of East Anglia down the hall from where Lou Kunkel (Norwich, England) and PhD in medical and Tony Monaco were identifying the Published online ahead of print. Publication date genetics under the tutorage of Malcolm Duchenne muscular dystrophy gene and available at www.jasn.org. Ferguson-Smith at the University of a couple of floors below where Stu Orkin Correspondence: Dr. Peter C. Harris, Division of Glasgow (Glasgow, Scotland) certainly was identifying the chronic granuloma- Nephrology and Hypertension, Mayo Clinic Roches- ter, 200 First Street SW, Rochester, MN 55905. piqued my interests in Mendelian disor- tous disease gene, the first successes of Phone: 507-266-0541; Fax: 507-266-9315; E-mail: ders. My interests were heightened by the positional cloning.1,2 [email protected] era; this was the time when positional In 1985, I first became aware of poly- Copyright ᮊ 2009 by the American Society of cloning (it was called reverse genetics cystic kidney disease (PKD), specifically Nephrology

1188 ISSN : 1046-6673/2006-1188 J Am Soc Nephrol 20: 1188–1198, 2009 www.jasn.org SPECIAL ARTICLE start of a long and fruitful collaboration Portuguese family showed the transloca- remarkably, immediately tail-to-tail to characterize the Oxford ADPKD pop- tion breakpoint was in the PKD1 candi- with TSC2, the polyadenylation sites just ulation; however, the first few years in date region and that individuals with the 60 bp apart.7,8 Because the majority of Oxford were less focused on PKD and balanced exchange had PKD, whereas the gene lay within the duplicated area, more on characterizing the vagaries of the son, missing the derivative chromo- the initial publication described only human telomeres and their role in chro- some containing the tip of 16 and part of partial sequence and a handful of muta- mosome rearrangements.4,5 22, had TSC. Since this family promised tions to identify it as the disease gene. It to pinpoint the location of PKD1 and to was not for another year that the full- help find the TSC gene, TSC2, we formed length sequence was revealed by amplifi- AUTOSOMAL DOMINANT a Consortium of European Groups (also cation of the gene from a radiation hy- POLYCYSTIC KIDNEY DISEASE involving Martijn Breuning’s and Dicky brid containing the tip of 16p (including Halley’s groups from the Netherlands) PKD1) but not the PKD1-pseudogenes PKD1 Gene Identification for these purposes. A consortium like located more proximally on 16p9 (Table ADPKD was pulled sharply back into fo- this with very focused and specific goals 1; Figure 1). The 5Ј end of the gene to cus following a call in 1992 from a med- can be remarkably productive; certainly exon 33 lay within the duplicated area ical geneticist from Cardiff, Julian Samp- these were very exciting, if nerve-wrack- with greater than 98% identity between son, describing a Portuguese family ing, times. The region was rapidly cloned PKD1 and the pseudogenes. We now segregating a :22 trans- as cosmids, mapped and characterized, know from completion of the Human location with PKD and a second domi- and genes were sought by hybridization Genome Project that there are six pseu- nant disease, tuberous sclerosis (TSC). to cDNA libraries (Table 1). Remarkable dogenes (PKD1 P1 through P6) that are Although ADPKD seemed to be the per- progress was made with the hard work in variously rearranged relative to PKD1. fect disease for positional cloning, many Oxford of Chris Ward, Jim Hughes, They express transcripts but seem to large families available and clear diag- Belen Peral, and our consortium part- have early mutations that prevent large nostics possible by imaging in adults, the ners. A number of large deletions identi- protein products from being generated.7 lack of informative recombinants, and fied the TSC2 gene.6 PKD1 itself was The second ADPKD gene, PKD2, was the gene-rich subtelomeric region that identified as a large gene intersected by identified by Stefan Somlo’s group in PKD1 lay in made identification of the the translocation breakpoint that lay 1996 with homology between the pre- gene problematic. Initial analysis of the within a complex duplicated area and, dicted PKD1 and PKD2 (poly-

Table 1. Comparison of phenotypes, gene identification, and characteristics of the genes/proteins in three forms of PKDa Disease Parameter ADPKD (PKD1) ARPKD MKS (MKS3) Typical presentation Adult In utero/neonatal/childhood In utero Phenotype Slowly progressive PKD (ESRD 54 yr) Rapidly progressive PKD Renal cystic dysplasia Liver cysts Congenital hepatic fibrosis Encephalocele Increased prevalence of ICA Biliary dysgenesis Polydactyly (rarely) Gene identification Genetic linkage human families Genetic linkage PCK rat/humans Genetic linkage wpk rat/human Gene pinpointed by chromosomal Genome sequence from HGP Genome sequence from translocation Gene identification (exon HGP Clone/map genomic region prediction, RT-PCR, Northern) Genes identified and (cosmids) Screen for mutations (DHPLC) annotated by HGP Screen/characterize genes (cDNA Screen for mutations (direct libraries, Northern analysis) sequencing) Screen for mutations (FIGE/RT- PCR) Gene characteristics 46 exons; 50 kb; ORF 12,909 bp 67 exons; 472 kb; ORF 12,222 bp 28 exons; 62 kb; ORF 2985 bp Exons 1 through 33 in reiterated region Protein characteristics 4303 aa; 460 kDa; 11 TM domains 4074 aa; 447 kDa; 1 TM domain 995 aa; 112 kDa; 7 TM Receptor-like protein Receptor-like protein domains Receptor-like protein Protein role Flow detector on cilia Required for normal cilia structure Cilia/ protein PCP associated protein? aaa, amino acids; HGP, Human Genome Project; RT-PCR, reverse transcription–PCR; TM, transmembrane; ICA, intracranial aneurysms; ORF, open-reading frame; FIGE, field-inversion gel electrophoresis.

J Am Soc Nephrol 20: 1188–1198, 2009 Gene Discovery in PKD 1189 SPECIAL ARTICLE www.jasn.org

ADPKD ARPKD MKS

Signal Leucine sequence rich repeats NH WSC domin 2 NH Signal sequence 2 LDL-A C-type Meckelin related lectin PA14 domain Polycystin-1 Signal PKD sequence NH2 Plus 8 other repeat TIG domains Cysteine rich proteins repeat

TMEM2 REJ module Polycystin-2 homology PROTEINS Transmembrane TRP channel GPS region homology Transmembrane Transmembrane domain region region

COOH COOH PLAT G-protein EF hand domain NH binding COOH 2 COOH

50 kb

1 510 1520 2530 354046 3 kb 1 10 20 30 40 50 60 67

1 10 20 28 PKD1 gene PKHD1 gene 5 kb MKS3 gene 1 kb 1 kb 10 20 30 40 50 60 67 PKD1 transcript 1102028 3 kb 250 bp 2 3 4 5 6 7 8 9 10 11 12 13 14 15 GENES 1 PKHD1 transcript MKS3 transcript

PKD2 gene Plus 8 other genes 1 kb PKD2 transcript

MKS3 mutations 19 MKS3 alleles 38 MKS1 mutations 14 MKS1 alleles 97 PKD1 mutations 430 PKD1 families 540 CEP290 mutations 15 CEP290 allels 24 PKD2 mutations 115 PKD2 families 200 Total mutations 300 Total alleles 680 CC2D2A mutations 1 CC2D2A alleles 22 Total mutations 545 Total families 740 Other mutations 16 Other alleles 22

MUTATIONS Total mutations 65 Total alleles 203

Strong genic effects Strong genic effects Strong allelic effects Strong allelic effects Weak allelic effects Genetic background important Genetic background/modifier Genetic background important loci important GENOTYPE PHENOTYPE CORRELATIONS

Figure 1. Summary of the genes, proteins, described mutations, and genotype/phenotype correlations associated with ADPKD, ARPKD, and MKS (only the MKS3 gene and protein are shown). Details of the mutations come from the ADPKD Mutation Database18 (ADPKD), ARPKD Mutation Database74 (ARPKD), and various articles describing MKS mutations. cystin-1 and Ϫ2), highlighting it from PKD1.13,14 Around this time, I was recruited cation mutations account for approxi- others in the candidate region.10 by Vicente Torres to Mayo Clinic. Torres’s mately 4% of cases.17 Both PKD1 and great knowledge of PKD plus his enthusiasm PKD2 are marked by extreme allelic het- Molecular Diagnostics and and assembled Mayo PKD population have erogeneity with even the most com- Genotype/Phenotype Correlations made this a very productive collaboration. monly described mutations (PKD1: in ADPKD Several full screens of PKD1 and PKD2, in- 5014delAG; PKD2: R306X and R872X) Molecular diagnostics was hampered by the cluding those spearheaded by Sandro Ros- individually representing only a little complex genomic region encoding PKD1, setti at Mayo, have now been described, with more than 1% of the ADPKD popula- and, in the first few years, the focus for muta- a clinical molecular test available.15,16 In a tion. A database of ADPKD mutations is tion screening was PKD2 and the single-copy clinically derived population, approximately hosted at Mayo, and details of described exons of PKD1 (34 through 46).11,12 Eventu- 85% of families have PKD1 and approxi- mutations are shown in Figure 1.18 Ap- ally, methods exploiting the rare base-pair mately 15% have PKD2.16 Base-pair muta- proximately 70% of ADPKD mutations differences between PKD1 and PKD1 P1 tions have been detected in up to 87% of are predicted to truncate the protein, through P6 allowed specific amplification of patients, whereas larger deletion/dupli- with 30% in-frame, mainly missense

1190 Journal of the American Society of Nephrology J Am Soc Nephrol 20: 1188–1198, 2009 www.jasn.org SPECIAL ARTICLE variants. Algorithms have been devel- gression. In CRISP, PKD1 kidney vol- Polycystin-2 localizes to the endoplasmic oped to differentiate pathogenic substi- umes (corrected for age and gender) reticulum, where it may modulate levels ϩ tutions from neutral variants.16 Dele- were almost twice the size of PKD2 (1041 of intracellular Ca2 and the primary ci- tions that disrupt PKD1 and the adjacent versus 623 ml),30 although there was no lium.37,38 It is thought to complex with TSC2 result in a contiguous gene syn- significant difference in the rate of its bigger brother polycystin-1. Polycys- drome characterized by various TSC growth of the kidneys (5.68%/y for tin-1 has 11 transmembrane domains phenotypes plus more severe renal cystic PKD1; 4.82%/y for PKD2; P ϭ 0.24). We (the final six homologous to polycystin- disease than found in PKD1 (or TSC2) therefore tested whether the number of 2), a large extracellular region, and a alone.17,19,20 cysts was significantly different and short cytoplasmic tail (Figure 1).9,39 The Knockout models indicate that in utero counting cyst number from a single mag- extracellular region consists of a number cyst development is associated with com- netic resonance slice showed that PKD1 of domains associated with protein–pro- plete loss of polycystin-1 or Ϫ2, with evi- kidneys had more cysts than PKD2 (31.5 tein or protein–carbohydrate interac- dence also from ADPKD cyst linings that versus 17.0; P Ͻ 0.001). This was espe- tions, 16 copies of a novel lg-like fold somatic mutation is important for cysto- cially true at a young age, suggesting that (PKD domain), and a receptor egg jelly genesis—a two-hit model of disease.21–23 PKD1 kidneys are larger because more (REJ) domain with homology with the However, cyst development in hypomor- cysts develop earlier. REJ family of sea urchin proteins.40–42 phic Pkd1 models expressing Ͻ20% of The role that allelic effects play in AD- Cleavage of the protein occurs at the G normally spliced product plus cysts in PKD is less clear. Mutation type is not protein coupled receptor proteolytic site overexpressing transgenics suggest that the associated with the severity of disease in (GPS) domain, and cleavage and translo- relative dosage of the polycystin protein PKD1 or PKD231,32; however, the posi- cation of part of the C-tail to the nucleus may also be important.24–26 Recently, we tion of the mutation in PKD1 (5Ј or 3Ј)is has also been described.43–45 Although identified two consanguineous families associated modestly with the severity of polycystin-1 has been localized to sites of homozygous for likely pathogenic mis- disease and propensity to develop intra- cell–cell and cell–matrix interaction, the sense changes.27 These variants in the het- cranial aneurysms, although the mecha- primary is likely a functional site erozygous state are associated with the de- nism is unclear.31,33 ADPKD displays a associated with PKD (see next section).38 A velopment of a few cysts, suggesting that wide range of intrafamilial phenotypic total of five human polycystin-1–like and they are incompletely penetrant alleles. In- variability. In rare cases, mosaicism or three polycystin-2–like proteins are heritance of such an allele in trans with a hypomorphic alleles may contribute to known. PKD1L3 and PKD2L1 encode the truncating PKD1 mutation is associated extreme intergenerational differenc- sour taste receptor, whereas PKDREJ is in- with early-onset ADPKD, suggesting these es,17,27,34 but it is likely that genetic back- volved in reproductive selection.46,47 alleles can significantly modify the pheno- ground accounts for a significant degree typic expression of the disease and high- of this variability. Recent progress in the ADPKD Is a light that the dosage of functional polycys- development of high-resolution single- Various pieces of data indicate that PKD tin may be relevant in human ADPKD.27 nucleotide polymorphism arrays will al- is associated with defects of the primary Gene type is a major determinant of low genome-wide studies to look for as- cilium. The primary cilium is a hair-like disease severity in ADPKD, with PKD1 sociations between common variants organelle rooted in the mother centriole associated with significantly more severe and the severity of disease in large, well- (the basal body) that projects from the disease than PKD2 (average age at ESRD characterized ADPKD populations. surface of the cell and is thought to have a of 54.3 yr compared with 74.0 yr).28 The sensory function.48 Caenorhabditis el- Consortium of Radiologic Imaging ADPKD Proteins Polycystin-1 and egans homologs of PKD1 and PKD2 are Studies of PKD (CRISP) monitored early ؊2 involved in male mating behavior and disease progression in 241 patients with The major outcome from finding a dis- specifically localize to cilia of sensory ADPKD by yearly determination of total ease gene is identification of the primary neurons.49 The Chlamydomonas or- kidney and cyst volume by magnetic res- defective protein. As is the case with AD- tholog of the protein defective in the onance imaging. During a 3-yr period, PKD and the polycystins, this can lead to Tg737 model of PKD (IFT88) is involved volumes were found to increase at an ex- the identification of a hitherto unknown in cargo transport within cilia/flagella ponential rate, with an average 5.27% in- protein family. Polycystin-2 is a distant (intraflagella transport), and the Tg737 crease in kidney volume per year.29 Pa- member of the transient receptor poten- mouse has shortened renal cilia.50–52 tients with the largest volumes (Ͼ1500 tial (TRP) family of ion channels, TRPP2 Conditional inactivation in the collect- ml) had a significant decrease in GFR (Figure 1).10,35,36 As with other TRP ing duct of the intraflagella transpor ki- (4.33 ml/min per yr), indicating that channels, it has six transmembrane do- nesin II motor subunit KIF3A results in larger volumes are emblematic of more mains and is thought to act as a homo- or progressive cyst development after birth; severe disease. Several clinical trials now heterotetramer. It is a nonselective cat- cystic cells lack primary cilia.53 Cilia may use change in renal volume as the pri- ion channel that transports Ca2ϩ and is act as flow sensors in kidney tubules with mary end point to monitor disease pro- modulated by intracellular Ca2ϩ levels. increased flow associated with a Ca2ϩ in-

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Ϫ Ϫ flux into the cell.54 Analysis of Pkd1 / log was also effective at inhibiting cystic cance of these alternative forms are avail- cells—and with an antibody blocking growth in both the liver and the kidney in able.71 polycystin-2—indicates that the polycys- an animal model and in preliminary data tin complex on the cilium acts as a flow from humans.65,66 Several additional po- Mutation Screening and Genotype/ sensor and that loss of either protein tential therapies are likely to enter clini- Phenotype Correlation abolishes the flow-dependent Ca2ϩ in- cal trials in the next few years. Details of described mutations are flux occurring through the polycystin-2 shown in Figure 1, with one mutation of channel.55 likely European origin, T36M, account- ing for approximately 17% of mutant al- Cellular Changes in PKD and AUTOSOMAL RECESSIVE leles.72–75 In contrast to ADPKD, only Prospects for Therapy POLYCYSTIC KIDNEY DISEASE approximately 40% of mutations are Consistent with the polycystin complex predicted to truncate the protein with ϩ having a role in Ca2 regulation, PKD Identification of PKHD1 approximately 60% in-frame (mainly cells display altered intracellular Ca2ϩ Our interest in the mainly infantile form of missense). There is little evidence of ge- homeostasis.56 In addition, increased PKD, ARPKD, came from the study of a rat netic heterogeneity in typical ARPKD, levels of cAMP seem to be a characteristic model of PKD, PCK. This recessively in- but genotype/phenotype correlations re- of PKD cells that may be directly related herited model, the result of a spontaneous lated to allelic events are clearly evident. ϩ to changes in Ca2 homeostasis.57–59 In mutation, has progressive cystic disease of All described cases with two truncating PKD cells, cAMP stimulates MAPK/ERK both kidney and liver.67 In this case, an en- mutations have the most severe disease, signaling compared with an inhibitory thusiastic nephrology fellow/PhD student, dying by the neonatal period with respi- response in normal cells.60,61 As well as Marie Hogan, mapped the gene in a cohort ratory problems.72,73 Viable cases have at activating the MAPK/ERK pathway and of 469 F2 animals in 2001.68 The region on least one missense change, indicating cell proliferation in PKD cells, cAMP rat chromosome 9 is syntenic to the human that many missense changes are incom- stimulates chloride-driven fluid secre- chromosome 6 interval where the ARPKD pletely penetrant alleles that presumably tion. Another change consistently found gene, PKHD1, had been mapped. Al- generate some functional protein. These in PKD cells is upregulation of mamma- though PKHD1 was initially mapped to a findings contrast with rodent models of lian target of rapamycin (mTOR). It has chromosome region in 1994, precisely lo- ARPKD where complete inactivation, al- been suggested that tuberin, the TSC2 calizing it and identifying the gene was though associated with liver disease sim- protein (that is a regulator of mTOR), hampered by the small size of families with ilar to human ARPKD, generally results and polycystin-1 interact.62 Develop- ARPKD, with few multiplex families (lack in only mild renal disease compared with ment of more severe disease when both of recombinants), and because the se- the dramatic in utero cystic expansion as- PKD1 and TSC2 are disrupted by contig- quence of the candidate region was not sociated with complete protein loss in uous deletion suggests cross-talk be- completed by the Human Genome humans.76,77 Further natural history tween the polycystin-1 and tuberin sig- Project.69,70 The greater genetic power of studies of human ARPKD showed that, naling pathways. the rat cohort coinciding with the initial although in utero–onset disease with A wide range of potential therapies posting of the candidate region genomic massive renal enlargement is typical, pre- have shown promise in PKD animal sequence in 2001 enabled Hogan and Chris sentation later in childhood or even as models. These target some of the cellular Ward to localize the gene to a 470-kb re- adults is relatively common.78,79 In these changes associated with PKD, including gion.68 Northern analysis in mouse with cases, kidney enlargement is typically less increased proliferation and altered fluid EST clones flanking the region demon- marked, with complications of portal hy- secretion (for review, see reference63). strated a large gene (13-kb transcript) pertension and bile duct dilation more Those that are already in Phase 3 clinical spanned the region and was hence a strong often the presenting phenotype. trials include a range of mTOR inhibitors PKHD1 candidate. Using an reverse tran- effective in animal models.62 In addition, scription–PCR approach linking predicted Fibrocystin, the ARPKD Protein patients who had ADPKD and under- exons, Ward expertly and rapidly cloned Fibrocystin is a large type 1 membrane went transplantation and received the entire human transcript and Rossetti protein with a short cytoplasmic tail and mTOR inhibitors as immunosuppressive screened a few hastily collected patients extensive extracellular region (Table 1; agents saw a size reduction of the native with ARPKD to identify the first muta- Figure 1).68,71 Similar to polycystin-1, a kidney and liver.62,64 The other agents in tions; hence, PKHD1 was identified (Table significant proportion of the extracellu- Phase 3 trials are those targeting the 1; Figure 1). There has been considerable lar region consists of repeats (12) of an heightened cAMP levels in PKD. This in- speculation about alternative splicing as a lg-like fold, this time the TIG/IPT do- cludes vasopressin receptor antagonists result of identification of multiple reverse main. One PKHD1 homolog, PKHDL1 that were effective at limiting renal cystic transcription–PCR products and prob- (encoding fibrocystin-L), that shows ho- disease in three animal models.57,58 Oct- lematic Northern analysis in humans (not mology over almost the entire length of reotide, a long-acting somatostatin ana- mouse), but little data about the signifi- the protein but is not associated with

1192 Journal of the American Society of Nephrology J Am Soc Nephrol 20: 1188–1198, 2009 www.jasn.org SPECIAL ARTICLE

ARPKD, has been identified.80 Fibrocystin tone) refined the interval to a 0.6-cM, ciliary defects (Figure 2).48 The connect- was found in the same complex as polycys- 2.05-Mb region.93 The syntenic interval ing cilium in the eye is essential for for- tin-2 and localized to cilia and the basal contained 13 conserved genes, and se- mation of the outer segment, and disrup- body.81–86 Interestingly, loss of fibrocystin quence analysis by two summer students, tion leads to retinal defects. The cilium, is associated with shorter cilia and struc- Erin Goranson and Stacie Lilliquist, un- although an important antenna detect- tural abnormalities.77,87,88 Furthermore, der the guidance of Mark Consugar, ing flow, is also essential for the function these cilia are often surrounded by small identified a nonconservative substitu- of several developmental pathways, in- vesicles (approximately 100 nm), whereas tion (P394L) in one (LOC313067). At the cluding Hedgehog (Hh) and Wnt. De- these vesicles are only rarely seen in wild- same time, Colin Johnson localized an fects in the Hh pathway are associated type mice.77 Mark Knepper’s group has MKS locus, MKS3, to a 12.5-Mb region with digital and craniofacial abnormali- highlighted that vesicles, including exo- that was syntenic to the Wpk candidate ties, as seen in several of these disor- somes from the multivesicular body path- region (8q21–22). Given the phenotypic ders.101–103 The PKD-associated protein, way, are common in urine and can be a similarity between MKS and wpk, we in- inversin, is a pivotal switch between the source of renal proteins as disease biomar- formed Johnson of our candidate, and, canonical and noncanonical Wnt path- kers.89 Polycystin-1 was identified as one on screening, he found mutations in five ways.104 The noncanonical pathway, also such exosomal protein, and we speculated families.93 A second MKS gene, MKS1, known as planar cell polarity (PCP), is the vesicles seen in the ARPKD model may was described in the same issue of Nature important for organizing polarity across be urinary exosomes. Ward and Hogan Genetics, which is particularly common a cellular layer. PCP plays a role in neural have now shown that a fraction of urinary in Finland.94 tube closure and hence may be involved exosomes are enriched not only for poly- in the exencephalic phenotypes found in cystin-1 but also polycystin-2 and fibrocys- Genetic Complexity of MKS MKS.105 PCP is also important for tubule tin.90 An analysis of these PKD exosomes Follow-up genetic studies by Consugar formation by orienting the mitotic spin- identified a specific proteome partially in mainly nonconsanguineous US and dle so that division occurs along the overlapping with previously described to- Dutch families showed that both MKS1 length of the tubule. Misorientation of tal exosome and total urine proteomes. and MKS3 are a common cause of MKS the spindle has been seen in PKD animal Cleavage of the in vivo polycystin-1 (at the in these populations (Figure 1).95 MKS3 models, suggesting that defects in PCP GPS cleavage site) and fibrocystin (at a is only rarely associated with polydactyly are a cause of tubule dilation as is seen in proprotein convertase site) has been dem- and in some cases with a milder CNS diseases such as ARPKD.106 onstrated. In vitro studies indicate these phenotype. Subsequent genetic studies PKD exosomes interact with cilia and may worldwide have shown a total of nine play a role in the flow response or a wider genes associated with MKS (reviewed in CONCLUSIONS role in urocrine signaling. reference63) (Figure 2). Additional ge- netic complexity has been shown with Illustrated here are three examples of hypomorphic MKS3 alleles associated PKD whereby identification of the dis- MECKEL SYNDROME with the viable ,96 ease gene was the first step toward un- whereas MKS1 mutation is linked with derstanding pathogenesis. Completion Identification of MKS3 Bardet-Bidel syndrome97 (Figure 2). In- of the Human Genome Project, atypi- The identification of a third PKD gene deed, it is now clear that a group of syn- cal patients, and animal models have occurred in the modern era of complete dromic disorders associated with PKD, greatly aided gene identification. Iden- human genomic sequence and full gene plus CNS, digital, and/or eye defects are tification of the disease gene has also annotation (Table 1). In this case, the related not only by overlapping pheno- aided molecular diagnostic that is now disease was Meckel syndrome (MKS; or types but also by considerable genetic routinely available in many of these Meckel-Gruber syndrome), a recessively overlap. Allelic effects may explain some disorders. A surprising finding is the inherited, lethal syndrome characterized of the phenotypic differences associated likely relatedness of PKD pathogenesis by renal cystic dysplasia, occipital en- with mutation to the same gene, but oligo- in different forms of the disease: That cephalocele, polydactyly, and biliary dys- genic inheritance and genetic background, defects in ciliary function are likely genesis. Our interest in this disease came including modification by hypomorphic central to pathogenesis. It is of note again from a rat model of PKD, wpk, that alleles, also seem important.97–100 that discoveries in primitive model sys- was first described in 2000 and further tems such as Chlamydomonas and C. characterized by Vince Gattone to have a Syndromic Forms of PKD Are elegans, plus studies of rodent models, central nervous system (CNS) defect of revealed the central role of cilia in these agenesis of the corpus callosum.91,92 The Localization of the proteins in these syn- disorders. New protein families, the Wpk gene was already known to lie on rat dromic disorders to the basal body polycystins and fibrocystins, have been chromosome 5, and analysis of 566 F2 and/or cilium likely explains the range of identified, and the normal role of these animals (as a collaboration with Gat- phenotypes as they are associated with proteins is now being elucidated. Un-

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PKD1 Autosomal Dominant Liver Polycystic Kidney Disease cysts PKD2 (ADPKD)

Autosomal Recessive PKHD1 Polycystic Kidney Disease (ADPKD)

Nephronophthisis NPHP2, 4, 5, 7–9 (NPHP) and NPHP3 Senior Loken Syndrome (SLS) NPHP1 Liver fibrosis Developmental AHI1 Joubert Syndrome and delay PKD, Related Disorders cystic ARL13B (JSRD) Cerebellar vermis dysplasia MKS3 hypoplasia Retinal CC2D2A CEP290 defects RPGRIP1L Meckel Syndrome NPHP3 Encephalocele (MKS)

BBS2 BBS6 BBS4 MKS1

Bardet Biedl Syndrome Cognitive Digital BBS1 BBS3 (BBS) impairment defects BBS5 BBS7–12 Obesity

CNS abnormalities Orofacial Digital Syndrome Facial/oral OFD1 (OFDS) defects

Figure 2. Summary of genes (left) and phenotypes (right) associated with various forms of PKD. A wide range of genetic and phenotypic overlap is seen in the syndromic diseases. derstanding their function and the cel- Gitomer, Jared Grantham, Matt Griffin, Lisa port of this work and colleagues at Mayo and Ox- lular changes associated with PKD has Guay-Woodford, Cynthia Hommerding, Katha- ford for constant support. revealed multiple points where thera- rina Hopp, James Ireland, Bernard King, Vickie peutic intervention may be possible. Kubly, Sumedha Kumar, Donna Lager, Nicholas Many such interventions have been LaRusso, Amanda Leightner, Luca Manganelli, DISCLOSURES shown to be effective in animal models, Anatoliy Masyuk, Tatyana Masyuk, Catherine None. and several have progressed to clinical Meyers, Phil Miller, Dawn Milliner, Steve trials. The prospects for effective ther- Mooney, Albert Ong, Priyanka Patel, York Pei, apies look infinitely brighter than Ron Perrone, Dorien Peters, Justin Peters, Lynn when we started down the road of gene Pritchard, Rachaneekorn Punyashthiti, Qi Qian, REFERENCES identification more than 20 yr ago. Stephanie Richardson, Dick Sandford, Robert Schrier, Tam Sneddon, Dorothy Spencer, Lana 1. Monaco AP, Neve RL, Colletti-Feener C, Bertelson CJ, Kurnit DM, Kunkel LM: Isola- Strmecki, Blagica Tanaskovska, Sandra Thomas, tion of candidate cDNAs for portions of the ACKNOWLEDGMENTS RoserTorra,Han-FangTuan,DeniseWalker,An- Duchenne muscular dystrophy gene. Na- gela Wandinger-Ness, Yu Wang, Shelly Whelan, ture 323: 646–651, 1986 Apart from those named specifically in the text, I Andrew Wilkie, Phomphimon Wongthida, John 2. Royer-Pokora B, Kunkel LM, Monaco AP, Goff SC, Newburger PE, Baehner RL, Cole thank the many skilled and highly motivated sci- Woollard, Wai Chong Wong, Eric Wu, David FS, Curnutte JT, Orkin SH: Cloning the entists whom I have worked and collaborated with Yuan, Klaus Zerres, and Jing Zhou. In addition, I gene for an inherited human disorder— over the past 25 years. These include Magdalana thank the many patients who have taken part in chronic granulomatous disease—on the Adeva, Richard Aspinwall, Bob Bacallao, Kyong- our various studies and the referring physicians basis of its chromosomal location. Nature tae Bae, Jason Bakeberg, Nicki Barton, William and genetic counselors. Finally, I thank National 322: 32–38, 1986 3. Reeders ST, Breuning MH, Davies KE, Ni- Bennett, Sarah Blair-Reed, Jim Bost, Arlene Chap- Institute of Diabetes and Digestive and Kidney cholls RD, Jarman AP, Higgs DR, Pearson man, Dominique Chauveau, Eliecer Coto, Peter Diseases, PKD Foundation, Mayo Foundation, PL, Weatherall DJ: A highly polymorphic Czarnecki,BrianDawson,VickiGamble,Berenice MRC, and the Wellcome Trust for financial sup- DNA marker linked to adult polycystic kid-

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