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Kidney International, Vol. 52 (1997), pp.33—38

Autosomal dominant polycystic disease with anticipation and Caroli's disease associated with a PKD1 mutation Rapid Communication

ROSER Toiu&, CELIA BADENAS, ALEJANDRO DARNELL, CoNcEPcIO BRi, ANGELS ESCORSELL, and XAVIER ESTIVILL

Nephrology Service, Service, Sonography Section of the Radiology Service, and Hepatology Service, Hospital ClInic, Barcelona, Spain

Autosomal dominant polycystic kidney disease with anticipation and (ESRD) than PKD1 patients [14]. The PKD1 transcript consists Caroli's disease associated with a PKD1 mutation. Autosomal dominant of 14,148 bp, distributed among 46 exons, spanning 52 kb. An polycystic kidney disease (ADPKD) is the most common renal hereditary interesting feature of this is that all but 3.5 kb at the 3'end disorder. Clinical expression of ADPKDshowsinterfamilial and intrafa- milial variability. We screened for mutations the 3' region of the PKD1 of the transcript is encoded by a region repeated several times, gene, from exon 43 to exon 46, in a family showing anticipation and proximally in the same chromosome. Until now very few muta- Caroli's disease and have found a 28 base pairs deletion in exon 46tions [15—19] have been reported in the PKDI gene, mainly due to (12801de128) and a new DNA variant in exon 43 (12184 C to G conserving the this fact, and most of them are located in the non-repeated Ala 3991) segregating with the disease. The mutation should result in a3'region. Only one of these mutations has been reported in more protein 44aminoacids longer than the wild-type PKD1. This PKDI mutation manifests as typical adult-onset disease in the father, but in the than one family [20]. proband, a 26-year-old man, ADPKD was diagnosed as a newborn and The presence of anticipation, defined as beginning of ESRD at was associated with Caroli's disease at the age of 18 years. A renal biopsy least ten years earlier in the offspring than in the parent or the performed in childhood disclosed a predominance of glomerular . Mutation 12801de128 is the first molecular defect associated with Caroli's presence of early-onset cases in a family, has been reported in disease and the PKD1 phenotype. The finding of the same mutation in two several ADPKD families [2, 14, 211. There is no molecular basis to different members of the same family with different expression of theexplain anticipation in ADPKD. The PKD1 gene has been fully disease indicates that the phenotypic variation in ADPKD must be due to sequenced and there are no trinucleotide repeats larger than five modifying factors that may radically affect the course of the disease. copies that could explain the phenomenon of anticipation as has been found in some neurological disorders [22]. Hepatic involvement is extremely frequent in ADPKD. It Autosomal dominant polycystic kidney disease (ADPKD) is theusually presents as polycystic disease (PLD), being more most common renal inherited disorder affecting 1 in 1000 personsfrequent as the age increases, and it is more severe in women [23, [1] and accounting for 10% of patients on renal replacement24]. PLD belongs to a family of liver diseases, characterized by an therapy (RRT) [21. It is characterized by the progressive renal cystovergrowth of biliary epithelium and supportive connective tissue, development and enlargement as well as a wide array of extrare-called the hepatobiliary fibropolycystic diseases [25]. These disor- nal features [31. Several reports reveal a high intrafamilial andders include dilation of the extrahepatic and/or intrahepatic bile interfamilial variability of the clinical expression of the disease [4,ducts, segmental dilatation of intrahepatic bile ducts (Caroli's disease), congenital hepatic fibrosis, autosomal recessive polycys- 51. At least three different may cause ADPKD: PKD1, intic kidney disease (ARPKD) and PLD. The common pathogenesis chromosome lopl3.3 [61 accounts for about 85% of the cases [7],for these entities may explain why the presence of these diseases PKD2at4q13 to 23 [8—10] accounts for the remaining 15% and afrequently overlaps. Hepatic fibrosis is always present in ARPKD third locus still unmappcd causes the disease in some reportedbut it is rare in ADPKD [26]; moreover, Caroli's disease is quite families [1 1—13]. PKD2 has been proved to be a milder form of the frequent in ARPKD [27, 28] but extremely infrequent in ADPKD disease with a later age of onset of end-stage renal disease[29—32]. Here we describe a deletion of 28 bp in the PKDJ gene in two members of the same family showing different expression of the disease: a severely affected young adult with ADPKD and Caroli's Key words: ADPKD, intrafamilial variability, hepatic disease, early onset, disease diagnosed as a newborn, and his father who has typical glomerular cysts. adult onset ADPKD. Received for publication January 25, 1997 and in revised form March 3, 1997 METHODS Accepted for publication March 3, 1997 Genomic DNAfrom150 unrelated individuals was extracted © 1997 by the International Society of Nephrology with the salting out method [33] and was amplified with several

33 34 Torra et al: PKD1 mutation, anticipation and Caroli's disease S i (I l.*-< Fig. 1. Renal biopsy of the proband at two months of age. Preserved renal tissue can be found among tubular and glomerular cysts. An

is"--- arrow points a glomerular .

primers that covered the DNA sequence from exon 43 to exon 46.ocrit 37%, platelets 39,000 per mm3 and prothrombin test 69%. The results in the present work were obtained with primers JJThe sonography scan disclosed with dilation of (12666-12848 nt of PKD1 eDNA; exon 46) using a DMSOintrahepatic bile ducts preferentially in the right lobe (Fig. 2), containing buffer and annealing at 60°C with 1.5 mi MgC12 andportal cavernomatosis, and cystic kidneys of 17 cm with primers DD (12084-12271 nt of PKDJ cDNA; exons 43 to 44) of diameter. Delayed views of a hepatobiliary scintigraphy 99mTc with the same buffer and PCR conditions as JJ [171. The productsDISIDA (diisopropyliminodiacetic acid) demonstrated retained of 183 bp and 263 bp, respectively, were analyzed by SSCA (single radiotracer, at 24-hours post-injection, preferentially in the right strand conformation analysis) with the silver staining techniquelobe. [341. The abnormal PCR fragments generated were sequenced The father is 53 years old and has polycystic liver and renal using ABI PRISM Dye Terminator Cycle Sequencing Readydisease. He has been on RRT for the last three months. The Reaction Kit in an ABI PRISM Genetic Analyzer 310 (Perkinmother and the brother (24-years-old) showed no evidence of Elmer, Foster City, KS, USA). Microsatellites AC25-D16S291,ADPKD either clinically or at . Two uncles were KG8-PKE1 and CW2-D16S663 were amplified and analyzed asdiagnosed with ADPKD and started RRT at the age of 60 and 58, previously described [14J. The Gene Bank accession number ofrespectively. The grandfather and the great-grandfather died at 58 the cDNA sequence used to number the nucleotides position isand 50 years of age, respectively, due to uremia. L33243. Molecular analysis RESULTS Linkage analysis using three microsatellites from the PKDJ region Clinical data (AC2.5-D16S291, KG8-PKD1 and CW2-D16S663) showed that the The proband is a 26-year-old man who was diagnosed as aparent had transmitted different alleles to his two sons (Fig. 3a). newborn of polycystic kidney disease due to palpation of enlarged The 3' end of the PKDJ genomic sequence of the father was kidneys. A renal biopsy, performed at two months of age, dis-analyzed for mutations by SSCA and an abnormal fragment was closed a renal surface covered by multiple cysts of 5 mm ofdetected with the primers JJ [17]. As the SSCA pattern suggested diameter. Microscopical analysis showed multiple cysts of aa deletion the PCR product was electrophoresed in a 4% agarose uniform size limited by cuboidal epithelium and separated bygel where two fragments of 183 and 155 bp could be seen in the scarce intercellular matrix in which renal tubuli and glomeruliaffected individuals and only one fragment of 183 bp in normal were embedded. There were also some cystic glomeruli (Fig. 1).subjects. Only the father and the affected son have the mutation He did not follow any medical control until the age of 18 years(Fig. 3b). Direct sequencing showed a 28 bp deletion (12801del28) when in a routine analysis pancitopenia was discovered. Panci-in exon 46 (Fig. 3c). This mutation induces a frameshift after topenia was attributed to portal diagnosed in hemo-amino acid 4200 and should result in a protein 44 amino acids dynamic studies. The cause of portal hypertension was supposedlonger than the normal PKDJ protein. The mutation 12801de128 to be either Caroli's disease associated with liver fibrosis or portalwas not detected in a further screen of 150 normal chromosomes. cavernomatosis, which were both present in the patient. At the Another abnormal fragment in the SSCA was found with age of 23 a slight deterioration of the renal function was discov-primers DD. Direct sequencing showed a C to G transition (12184 ered and a renal sonography disclosed enlarged cystic kidneys. C to G conserving Ala 3991) in exon 43. The same abnormal band At the time of the study, the creatinine was 4 mg/dl, thewas present in the affected son (Fig. 3a). This change was not seen creatinine clearance 20 ml/min, leukocytes 1400 per mm3, hemat-in a further study of 300 chromosomes. Ton-a et al: PKDI mutation, anticipation and Caroli's disease 35

-- — -a-I- Fig. 2. Sonography image of the right lobe of the liver showing tubular (up) and sacular t (down) dilation of the intrahepatic bile ducts.

DISCUSSION postulate than an abnormal polycystine may account for the ADPKD is a multisystem disease with abnormalities in manyabnormal biliary organogenesis and an early onset renal disease. different organs. The extent of the affection of these organs may There have been several reports of intrafamilial clinical heter- vary greatly causing a great interindividual variability. The age ofogeneity in ADPKD and anticipation [21, 5, 39], but there is only onset of ESRD is the parameter that is most widely used toone report disclosing the molecular defect in a case with antici- differentiate the clinical expression of the disease [21]. In thepation. Peral et al described a Y3818X mutation in a severely family presented in this study, not only the age of onset of ESRDaffected child. They found the same mutation in her clinically caused great intrafamilial variability but also the hepatic disease.normal twin brother and in her father who had typical adult-onset While the father has a typical PLD the son has a Caroli's diseasedisease [16]. Our analysis of one such family has revealed a with hepatic fibrosis. deletion of 28 bp in the 3' region of the PKD1 gene. This mutation Two varieties of Caroli's disease have been described [35]. Achanges the open reading frame and should produce a protein pure form characterized by ecstasies of the intrahepatic bile ductswith additional 44 amino acids. Some other mutations have been without further hystological abnormalities and a combined formdescribed in this region of PKDJ in families with adult onset in which Caroli's disease is associated with periportal fibrosisADPKD [15, 17—19]. In contrast to many neurological diseases corresponding to congenital hepatic fibrosis. The association ofwhere anticipation can be explained by the amplification of an both types of Caroli's disease and autosomal recessive polycysticunstable DNA repeat in the coding or noncoding region of the kidney disease (ARPKD) has been well recognized in the litera-relevant genes [22], there are no trinucleotide repeats larger than ture [27, 28, 36], but the coexistence of ADPKD and Caroli's5 units in length in the PKDJ gene. Thus, the anticipation seen in disease is a rarely reported association [29—32]. The pathogenesisADPKD is not due to a within the PKD1 gene. of Caroli's disease seems to involve a partial arrest of the Different explanations may account for the intrafamilial vari- embriogenesis of larger intrahepatic bile ducts (segmental ducts).ability seen in the family presented in this study. One possibility is The presence of hepatic fibrosis implies that the defect is not onlythat there may be subtle changes in the PKD1 gene inherited from present during the early stages of embriogenesis, but alsothe mother, which are not enough to cause the disease but may on a later stage of development of more peripheral biliaryinfluence the expression of the other allele and modify the ramifications (interlobular ducts) [361. The typical hepatic in-presentation of the disease. A two-hit model [40, 41] could also volvement in ADPKD which is PLD and microbiliary hamartomasaccount for the intrafamilial variability seen in this family with (von Meyenburg complexes) invokes a factor arresting or perturb-different and more severe somatic mutations in the normal PKD1 ing the remodeling of primitive biliary ducts in the later phases ofallele in the son than in the father. It is also possible that the the development (intralobular ductules). Thus, in the case pre-proband has a de novo mutation in the other allele or in the same sented here there is an arrest of the biliary embriogenesis muchPKD1 gene that is absent in the father. Eventually, modifying sooner than would be expected in a usual case of ADPKD.genes may be one of the best explanations for the clinical Polycystine, the product of the PKDI gene, seems to be involveddifferences between ADPKD patients. Some modifying genes in in both renal and hepatobiliary embriogenesis. This is stronglypolycystic mouse models affecting the progression of the disease supported by the finding that polycystine is more highly expressedhave been described [42, 43], but nothing is yet known in humans. in fetal than in adult kidney [371 and is also overexpressed in theThe PKD2 gene may be one of these modifying genes as its liver and the biliary fetal system [38], but it is highly speculative toproduct has been proved to interact with polycystine through a 36 Ton-aet al: PKDI mutation, anticipation and Caroli's disease Al 0—- — B 12* 111* 112 Ii bp 1 2 A5 Al A5 A5 12801/1 2802 del 28 — 12184C—+G 183— C7 C4 05 C3 155— F4 F13 F6 F5 1 II Al A5 A5A1

12801 /1 2802 del 28 12184 C—G C4 C3 05 04 F13 F5 F6 F13

C Wild-type gggGLScig age gtgVSLGRI.OTRCEPIP age ctg ggccggctggggacaaggtgt gag cct gag eecteeSRLQAV ege etc caa gee gIg tIclEALLTQFDRLNQATEDVYQI.lQgag gee etg ctc acecag Ittgac ega etc aaeeaggee aca gag gac gte tue cag etg gag eag eag etg cue age etg eaa gge ege agg age age egg gcg ccc gee gga tet tee egt ggc ecu tec ceg Q I. II SI. Q 0R R S S R A I' A 0 S S R G P S P ggcGLRetg egg ecuPAL gea ctg cccP age SRLARASROVI)I. ege ett gee egg gee agt egg ggt gIg gue ctg geeATOP act gge ccc agesRTPLRAKNKVHPSSTZ ugg aca ccc elI egg gee aug aae aug gte cue eec age age act tag

Mutant ggg ctg agt gIg age etg age ect cccgee tee aug ccg tgt leg agg eec tgc tea eec agt ttg ace C; I.SV SI.S PPASKPCSRPCSPSLT gactea ace agg ecu eag agg aeg telace age tgg age age age tge aea gee tgc aug gee gea gga I)S T kP Q K T STS W S S S C TA C K A A C geagee ggg ege ceg eeg gal cIt cccgIggee cat ccc egg gee tge gge cag cue ige ecu gee gee AA G K P I'I) L PVA HP RACGQHCI'AA hgccc ggg ecu gte ggg gtg tgg acetgg ecu etg gee ecu geu ggu cue Ccc ttc ggg ecu aga aeu L 1' 0 I' V C V W 1WP1 A I' A C H P F (; P K T aggIce ace ecu gca gea ett agt eetecttee tgg egg ggg tgg gee gIg gag teg gag tgg aca ceg K S1' P A A L S PPS WK C W A V E S F W T P etcagl alt act tie tge cgc tgt euaggeega ggg ecu ggc aga atg get gea egt agg tIe eec aga I SI'F CC R C Q(1 KC P C 'I'M AA K KF PK gagcag gea ggg gea tel gte tgt etgtggget tea gca ett taa FQ A C A S VCLWA S A I Z Fig. 3. A. Pedigree of the kindred. Symbols are: (0, U), unaffected; (•,affected;U, U, men; 0 females. Haplotypes from the PKD1 region are below each symbol: microsatellite alleles: A, KG8; C, AC2.5; F, CW2. Mutation and the new described polymorphism are also indicated. B. PCR amplification with primers JJ in which affected members show an heterozygous pattern, due to the deletion of 28 bp. C. Wild-type and mutant DNA sequence from position 12649 in exon 46 and predicted protein sequence given in the one letter code (accession L33243).The predicted protein from mutant DNA is 44 amino acids longer than the wild type. For the wild-type, the deleted DNA sequence is shown in bold; for the mutant, the aminoacid sequence changes caused by the mutation are shown in hold.

coiled coil domain [44]. Thus, subtle changes in the PKD2gene, went a renal biopsy at the age of two months. The most striking either de novo or inherited from the mother, could account for thefeature of this renal biopsy was a predominance of glomerular clinical differences seen in this family. Finally, since about 25% ofcysts. This finding has been reported in the literature in autopsies the general population have trinucleotide repeats of larger thanof newborns and in fetuses with ADPKD [45—49] and has been 40 units (X. Estivill and M. Pujana, personal communication), it isconsidered to be a common feature of early-onset ADPKD. possible that genes with trinucleotide repeats present expansionsAlthoughour patient will enter ESRD sooner than usual and this that influence the severity of ADPKD. may suggest that glomerular cysts imply a worse outcome, the Infantile renal tissue of ADPKD patients surviving the neonatalpresence of glomerular cysts could also be a common finding in period is rarely available but the patient reported hereby under-ADPKD kidneys in the early stages of development. This last Torra et at: PKD1 mutation, anticipation and Caroli's disease 37

hypothesis is supported by the fact that large cysts in adults can be 13. TURCO AE, CLEMENTI M, ROsETTI 5, TENCONI R, PIGNATFI PF: An more difficult to identify hystologically as being glomerular and its Italian family with autosomal dominant polycystic kidney disease unlinked either to the PKD1 or PKD2 gene. Am J Kidney Dis prevalence may be underestimated. 28:759—761, 1996 In summary, the family reported hereby discloses very different 14. TORRA R, BADENAS C, DARNELL A, NICOLAU C, VOL PINI V, REVERT phenotypes arising from the same mutation. Probably the coex- L, ESTIVILL X: Linkage, clinical features and prognosis of ADPKD istence of other genetic defects with this mutation may account for types I and 2. JAm Soc Nephrol 7:2142—215 1, 1996 the anticipation and/or the hepatic involvement seen in this 15. PERAL B, GAMBLE V, SAN MILLAN JL, STRONG C, SLOANE-STANLEY J, MORENO F, HARRIS PC: Splicing mutations of the polycystic kidney family. 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