Prenatal Diagnosis of Autosomal RecessivePolycystic Kidney Disease (ARPKD): MolecularGenetics, Clinical Experience, and Fetal Morphology

Klaus Zerres,1* Gabi Mucher,1 Jutta Becker,1 Carsten Steinkamm,1 Sabine Rudnik-Schoneborn,1Paivi Heikkila,2 Juhani Rapola,3 Riitta Salonen,4 Gregory G. Germino,5 Luiz Onuchic,5Stefan Somlo,6 Ellis D. Avner,7 Leigh A. Harman,8 John M. Stockwin,9 andLisa M. Guay-Woodford9,10

1Institute for Human Genetics, University of Bonn, Bonn, Germany2Department of Pathology, University of Helsinki, Helsinki, Finland3Children’s Hospital, University of Helsinki, Helsinki, Finland4Prenatal Genetics, Department of Obstetrics and Gynaecology, University of Helsinki, Helsinki, Finland5Department of Medicine, Johns Hopkins University, Baltimore, Maryland6Department of Medicine, Albert Einstein College of Medicine, Bronx, New York7Department of Pediatrics, Rainbow Babies’ and Children’s Hospital, Case Western Reserve University,Cleveland, Ohio

8Department of Immunogenetics/DNA Diagnostics Laboratory, University of Alabama at Birmingham,Birmingham, Alabama

9Department of Medicine, University of Alabama at Birmingham, Birmingham, Alabama10Department of Pediatrics, University of Alabama at Birmingham, Birmingham, Alabama

Autosomal recessive polycystic kidney dis-ease (ARPKD) is one of the most commonhereditary renal cystic diseases and has ahigh infant mortality. Prenatal diagnosisusing fetal sonography can be unreliable,especially in early pregnancy. The ARPKDlocus has been mapped to proximal chromo-some 6p allowing haplotype-based prenataldiagnosis in ‘‘at-risk’’ families. From Decem-ber 1994 to March 1997, we received 258 in-quiries regarding prenatal evaluation andwe have completed analyses in 212 families.To date, 65 prenatal analyses have been per-formed in 57 families. In the majority of therequesting families (45/57), the index chil-dren are deceased and their DNA was ex-tracted from paraffin-embedded tissue.Eighteen fetuses were homozygous for thedisease-associated haplotypes. In 12 of thesefetuses, pathoanatomical examination dem-onstrated typical ARPKD changes consist-ing of dilated collecting ducts and the char-acteristic hepatic ductal plate malforma-

tion. These changes were detected in twofetuses as early as 13 weeks gestational age.These cases represent the earliest demon-stration of ARPKD-associated histopathol-ogy reported to date. One high risk fetuswas carried to term and turned out to beunaffected. However, the diagnosis ofARPKD remained doubtful in the index pa-tient. Forty-three fetuses were either het-erozygous or homozygous for a nondisease-associated haplotype and all infants bornwere phenotypically unaffected at birth. Infour cases, a recombination event occurredbetween the flanking markers and no geno-typic prediction was possible. Three of thesepregnancies were terminated and necropsyof the fetuses confirmed ARPKD, while onefetus was carried to term and showed no ab-normalities at birth.

These results show that haplotype-basedprenatal testing is feasible and reliable inpregnancies ‘‘at risk’’ for ARPKD. An abso-lute prerequisite for these studies is an ac-curate diagnosis of ARPKD in previously af-fected sib(s). Am. J. Med. Genet. 76:137–144,1998. © 1998 Wiley-Liss, Inc.

KEY WORDS: autosomal recessive polycys-tic kidney disease; prenataldiagnosis; molecular genet-ics; fetal morphology

Contract grant sponsor: Deutsche Forschungsgemeinschaft;Contract grant sponsor: NIH; Contract grant number:R01DK51034.

*Correspondence to: Klaus Zerres, M.D., Institut fur Human-genetik der Universitat Bonn, Wilhelmstr. 31, D-53111 Bonn,Germany. E-mail: zerres@humgen.uni-bonn.de

Received 20 May 1997; Accepted 21 October 1997

American Journal of Medical Genetics 76:137–144 (1998)

© 1998 Wiley-Liss, Inc.

INTRODUCTION

The clinical spectrum of autosomal recessive polycys-tic kidney disease (ARPKD) is widely variable withmost cases presenting in infancy [Cole et al., 1987;Kaplan et al., 1989; Zerres, 1992; Zerres et al., 1996].Its principal manifestations involve the dilatation ofthe collecting ducts and dysgenesis of the portal triad.Typically, ARPKD patients are identified either inutero or at birth. Affected fetuses characteristicallyhave bilaterally enlarged echogenic kidneys and oftenoligohydramnios due to poor fetal urine output, whichusually is not manifest before 20 weeks of gestation. Asa result of the oligohydramnios, these infants developthe ‘‘Potter phenotype,’’ consisting of pulmonary hypo-plasia, a characteristic facies, and deformities of thespine and limbs. As many as 30–50% of affected neo-nates die shortly after birth due to respiratory insuffi-ciency. Hypertension, renal failure, and portal hyper-tension due to hepatic fibrosis are significant causes ofmorbidity in children surviving the neonatal period[Zerres et al., 1996]. Due to the poor prognosis of earlymanifestations of ARPKD, there is a strong demand forprenatal diagnosis.

Historically, prenatal diagnoses of ARPKD havebeen made either by screening fetal sonography or byserial sonograms in ‘‘high risk’’ pregnancies. However,fetal sonography has limited reliability, as renal en-largement and increased renal echogenicity is often notdetected until late in the second trimester [Luthy andHirsch, 1985; Zerres et al., 1988]. Recent studies havedemonstrated that ARPKD maps to 6p21.1-p12 and todate, there is no evidence of genetic heterogeneity[Zerres et al., 1994; Mucher et al., 1995; Guay-Woodford et al., 1995]. The candidate interval spansless than 4 cM which is flanked distally by a cluster ofmicrosatellite markers (D6S272, D6S465, D6S427,D6S436, D6S243, D6S1714) and proximally byD6S466. These linkage data and the absence of geneticheterogeneity in all families tested to date provide thegenetic basis for DNA-based prenatal analyses in ‘‘atrisk’’ pregnancies.

In this report, we present the results of 65 DNA-based prenatal diagnoses performed at the Institute forHuman Genetics, University of Bonn, and the Univer-sity of Alabama at Birmingham. Particular emphasis isgiven to the prerequisites and limitations for theseanalyses.

SUBJECTS AND METHODS

Between December 1994 and March 1997, 258 inqui-ries were received from 250 families for haplotype-based prenatal diagnosis of ARPKD. These familieswere primarily of European and American origin. Atotal of 65 prenatal analyses have been performed in 57families. The family structures in detail were: one af-fected child only (31), two affected children withouthealthy sibs (7), and families with one (15), two (2), andthree healthy sibs (2), of whom 6 had at least one fur-ther affected child in addition to the index patient.Among the 11 index patients who survived the perina-tal period, the age at diagnosis ranged from the mid-

second trimester to 5 10/12 years. However, for themajority of these affected children (7/11), the diagnosiswas established either prenatally or at birth. Clinicalfollow-up of these survivors ranged from 9 months to 7years. Both parents and at least one affected child wereanalyzed in all families. In case there were further sib-lings available, they were included in the linkage studyas well.

Diagnostic Criteria

Of the 57 families who underwent prenatal diagno-sis, the diagnosis was pathoanatomically proven in 43cases (criteria see below). The remaining patients ful-filled the following diagnostic criteria: (1) manifesta-tion of typical clinical signs with characteristic ultra-sonographic signs according to Garel [1984] or (2) pres-ence of at least one of the following criteria: (a) absenceof renal cysts in the ultrasound examination of bothparents (this criterion was fulfilled in all but five fami-lies where the diagnosis was pathoanatomicallyproven), (b) clinical signs or histopathologic evidence ofhepatic fibrosis, (c) pathoanatomical proof of ARPKD inan affected sibling, and (d) parental consanguinity sug-gesting autosomal recessive inheritance.

Available results of ultrasound examinations of pre-vious pregnancies with affected fetuses should alwaysbe taken into consideration. Oligohydramnios is un-usual before 20 weeks gestation in ARPKD and hasbeen described only in rare cases. The presence of dis-crete cysts in the fetal ultrasound cannot be observedin ARPKD.

The histopathological criteria used were: (1) kidney:(a) fetal kidneys—slender dilated collecting ducts radi-ally from the medulla to the nephrogenic zone whereactive nephrogenesis is evident; (b) neonatal kidneys—cystic change is confined to dilated tubules (diameter<2 mm) which are radially arrayed in the cortex in anorientation perpendicular to the renal capsule; (c) olderinfants/children—dilated and elongated cortical col-lecting ducts with large, ectatic medullary collectingducts; no evidence of glomerular cysts, and (2) liver:congenital hepatic fibrosis with portal and interlobularfibrosis and biliary duct hyperplasia [Guay-Woodfordet al., 1998].

DNA Extraction

DNA was obtained from all families under informedconsent. For living pedigree members, DNA was ex-tracted from peripheral blood lymphocytes. In the ma-jority of families, the affected children were deceasedalready and DNA was extracted from paraffin-embedded or stored tissue (110/187) using either acommercially available method (Oncor EX-WAX; On-cor, Gaithersburg, MD) as previously described [Guay-Woodford et al., 1995] or a method described by Egg-ermann et al. [1994], which was also used for DNAextraction from microscopic slides in a few cases.

Polymerase Chain Reaction (PCR)Amplification and Haplotype-Based Analysis

Microsatellite markers were selected from theARPKD interval (Fig. 1). All microsatellites markers

138 Zerres et al.

used in this study had 6–14 alleles and a heterozygos-ity frequency of 0.6–0.87 [for details see Dib et al.,1996]. Marker D6S243 demonstrated in our material aheterozygosity frequency of 0.87 which is in contrast tothe frequency of 0.53 reported by Orphanes et al.[1993]. Primer pairs were either synthesized usingnucleotide sequences available in the Genome DataBase (accessible at http://gdbwww.gdb.org) or pur-chased from Research Genetics (Huntsville, AL). Mi-crosatellite amplification from lymphocyte-derivedDNA was performed as previously described [Guay-Woodford et al., 1995]. For analysis of DNA extractedfrom paraffin-embedded material or microscopic slides,marker selection was biased toward those primer pairswhich yielded the shortest products. PCR amplificationwas performed as previously described [Guay-Woodford et al., 1995]. In several cases, even thosemarkers with short products could not be amplified andconsequently these families were not informative forfurther analyses.

RESULTSPrenatal Diagnosis

Among the 258 inquiries regarding requests for pre-natal diagnosis (total 250 families), 38 families wereexcluded due to diagnostic doubts (13), insufficientfamily material (23), or lack of available DNA from theaffected child (2). A total of 65 prenatal diagnoses wereperformed in 57 families. Due to a set of highly poly-morphic markers available, all analyzed families wereinformative for at least two flanking markers with theexception of two families. Fetal material was obtained

by chorionic villus sampling (CVS) in 45 pregnancies.Due to a more advanced gestational age, amniocentesiswas performed in 19 pregnancies and fetal blood sam-pling was done in one patient.

As shown in Figure 2, predictions based on the fetalgenotype were possible in 61 pregnancies. Forty-threefetuses were predicted to be unaffected. Of these, 41had no evidence of renal sonographic abnormalities atbirth. One pregnancy was terminated for an unrelatedreason (XXY karyotype), and a second trimester mis-carriage occurred in another pregnancy. Twenty-twofetuses were predicted to be either affected (18) or athigh risk for ARPKD (4) (see below). Sixteen of thesepregnancies were terminated and ARPKD was con-firmed by histopathology in all 12 cases where a patho-anatomical examination was undertaken. Five preg-nancies were carried to term and ARPKD was con-firmed either by necropsy (2) or renal ultrasound (1).One fetus predicted to be affected did not give evidenceof ARPKD at birth, further pathoanatomical studiesraised doubts about the correct diagnosis in the indexpatient (see below, family 4). Another pregnancy,where a recombination event had complicated the in-terpretation, was carried to term and resulted in anunaffected infant. One pregnancy was ended by a spon-taneous second trimester miscarriage.

Recombination Events

Among the 212 analyzed families, we found a total of10 recombination events between flanking markers inone parental haplotype. In 4 of the 65 prenatal diag-noses, the fetus shared a parental haplotype with theindex case, and a recombination event occurred withinthe critical ARPKD interval in the other parental hap-lotype. It could not be determined whether the fetuswas homozygous or heterozygous for the ARPKD mu-tation. Because our recombinational mapping data in-dicate that the ARPKD gene probably lies closer to thetelomeric flanking marker (data not shown), we pre-dicted that the fetus was at greater than a 50% risk forARPKD in all four pregnancies. One pregnancy wascarried to term and resulted in an unaffected infant.After additional counseling sessions, the remainingparents decided to terminate the pregnancy. Patho-anatomical examination confirmed the diagnosis ofARPKD in all three cases. Cases 1 and 2 have beendescribed previously [Guay-Woodford et al., 1995;

Fig. 1. Ideogram of human chromosome 6p, with an expanded geneticmap of the ARPKD region and the relative order of used markers. Therelative order of the markers in the box is not known. Sex average dis-tances are those published by Volz et al. [1994]. MUT, methylmalonyl-coenzyme A; TCTE, T-complex-associated-testes-expressed-1 gene;KRAS1P, Kirsten rat sarcoma 1 viral ocogene homologue.

Fig. 2. Results of the 65 prenatal diagnoses. W, wild-type; M, mutant;Rec, recombination between flanking markers.

Prenatal Diagnosis of ARPKD 139

Zerres et al., 1995; Wisser et al., 1995]; details of bothfamilies were as follows.

Families 1 and 2. In both families the fetusesshared the maternal haplotypes with the affected chil-dren. A paternal recombination occurred between theclosest informative telomeric markers D6S1714 (family1) and D6S243 (family 2), respectively, and D6S466(centromeric) in both families.

Family 3. The index case (Fig. 3, II, 1) was born withenlarged, polycystic kidneys and died within 24 hoursafter birth. Autopsy confirmed the diagnosis ofARPKD. In the second pregnancy (II, 2), amniocentesiswas performed at 14 weeks. Given the substantial riskthat the fetus was affected, the parents decided to ter-minate the pregnancy at 21 weeks gestation. Histo-pathologic analysis revealed dilatation of the collectingducts and the ductal plate malformation in the fetalliver. After a further pregnancy (II, 3) it could be de-termined that the recombination event occurred in thefirst affected child (II, 1).

Evaluation of Possible Genetic Heterogeneity

In addition to these 65 cases, six other families werereferred for haplotype-based prenatal ARPKD testing.A single index case in each family carried the diagnosisof ARPKD. In four families, the clinical course and/orthe autopsy reports raised suspicion about the diagno-sis. These concerns prompted careful review of the his-topathology from the putative index case prior to pre-natal analyses. The reevaluation excluded ARPKD asthe diagnosis and revealed that the renal cystic diseasein these index cases was most likely due to cystic dys-plasia, glomerulocystic kidney disease, or Meckel syn-drome.

Two other families had initially been accepted for alinkage analysis. In both families, haplotype data wereincompatible with the assumption of 6p linkage. Bothcases most likely are not ARPKD but illustrate the dif-

ficulties in making the diagnosis ARPKD and under-line that the most critical point in prenatal diagnosis isthe diagnosis of ARPKD in a previously affected child.

Family 4. The index child presented as a newbornwith enlarged kidneys and a few visible cysts on renalultrasound. Despite our concerns about a possible un-certainty of the diagnosis, prenatal diagnosis was re-quested by the obstetrician in agreement with the par-ents at 20 weeks gestation in a subsequent pregnancy.The fetus and the putative affected child shared thesame parental haplotypes. Nevertheless, the preg-nancy was carried to term and sonography did not re-veal any renal abnormalities in the neonate.

Family 5. Prenatal sonography at 21 weeks gesta-tion revealed enlarged kidneys with increased echo-genicity and fetal hydrops. The parents decided to ter-minate the pregnancy. Based on histopathological ex-amination, the diagnosis of ARPKD was made initially.The histopathology was initially interpreted as ‘‘com-patible’’ with ARPKD but not absolutely typical. Noother malformations were detected in the fetus. Ge-netic analysis demonstrated that the fetus and a 3-year-old asymptomatic son shared identical haplo-types. At this point, the renal and biliary pathologywere retrieved and reevaluated. The most notable re-nal findings were fine cortical cysts, involving both glo-merular and tubular structures, and rudimentary med-ullary pyramids. This histopathologic pattern is moreconsistent with either dominant polycystic kidney dis-ease or with sporadic glomerulocystic kidney diseasethan with ARPKD (J. Bernstein, personal communica-tion).

Early Morphological Findings

Besides the two mentioned cases with fetal changes,10 additional fetuses were terminated at ages 13 to 23weeks gestation who were examined pathoanatomi-cally. All fetuses revealed typical changes of ARPKD.The following two examples demonstrate the earliestso far described histological findings in affected fetusesof 13 and 16 weeks gestation.

Family 6. A first child died at the age of 3.5 hours.At autopsy, enormously enlarged kidneys were found(length 10 cm, weight 232 g/214 g) with typical cysticdilatation of collecting ducts in medulla and cortex. Theliver weighed 185 g and demonstrated typical congen-ital hepatic fibrosis. After birth of a healthy daughter,termination of a further pregnancy at 21 weeks of ges-tation was undertaken after sonographic detection oflarge echogenic kidneys. Typical pathological changeswere evident in the kidneys and liver. A fourth preg-nancy was terminated at 13 weeks of gestation afterDNA-based analysis predicted that the fetus was af-fected. Histopathological analysis revealed slightly di-lated medullary and cortical collecting ducts (Fig. 4a).In the liver, the typical ductal plate malformation wasevident with expanded portal areas that contained anincreased number of bile ducts. In a subsequent preg-nancy, prenatal diagnosis predicted that the fetus wasunaffected and a healthy girl was born.

Family 7. The index patient was noted to havegrossly enlarged kidneys in utero and the child subse-

Fig. 3. Pedigree of family 3 with recombination event between flankingmarkers (see text).

140 Zerres et al.

quently died 10 days after birth. Autopsy revealedhuge kidneys (219 g/235 g). Grossly, the cut surface hasa sponge-like appearance with cystic involvement of90% of the parenchyma. The liver (167 g) showed thetypical features of congenital hepatic fibrosis. DNA-based analysis in a subsequent pregnancy predictedthat the fetus was affected. Histopathologic analysisfollowing termination at 16 weeks gestation revealeddilated collecting ducts as well as a few discrete cysts,which are derived possibly from distal tubules (Fig. 4b).The characteristic hepatic ductal plate malformation

was evident. A third pregnancy was also terminatedbased on prenatal DNA analysis. Patho-anatomical ex-amination again revealed the typical changes ofARPKD in this fetus (Fig. 4c and d).

DISCUSSION

Despite the broad clinical spectrum of ARPKD, in-cluding rare mild cases resembling adult-onset autoso-mal dominant PKD, the clinical variability among af-fected siblings with ARPKD may be limited [Deget et

Fig. 4. Fetal morphology of ARPKD of three different fetuses (see text). A: Kidney histopathology in an affected fetus at 13 weeks gestational age(family 6). Note the dilated ducts from medulla to subcapsular area. H&E, ×62. B: Elongated cysts in the kidney at 16 gestational weeks seen in the firstfetus of family 7. H&E, ×62. C: Dilated collecting ducts from medulla to subcapsular area in a fetal kidney examined at 13 gestational weeks (second fetusof family 7). H&E, ×62. D: Ductal plate malformation of a hepatic portal tract of a fetal liver at 13 gestational weeks (second fetus of family 7). Note theincreased number of bile ducts at the border of the hepatic lobule and the portal area. H&E, ×62.

Prenatal Diagnosis of ARPKD 141

al., 1995]. Therefore, families with a severely affectedchild carry a high risk that additional affected childrenwill be severely affected as well. Even for those chil-dren with the severe perinatal phenotype, serial fetalultrasound is not reliable in detecting the disease. Inthis present study, five independent siblings at risk forARPKD were followed by serial sonographic examina-tion from the first trimester of pregnancy, before DNA-based prenatal prediction had become available. Thefetuses appeared to be entirely normal up to 20 weeksgestation and the diagnosis was first suspected be-tween 21 and 31 weeks gestation. These observationsare in accordance with previous reports [Luthy andHirsch, 1985; Zerres et al., 1988] and support the con-clusion that a reliable early prenatal diagnosis can onlybe offered by use of DNA-based methods.

Incidence and Heterozygosity Frequency

The exact incidence of ARPKD is not known. Figuresrange from 1:6,000–1:14,000 [Bosniak and Ambos,1975] to 1:55,000 [Potter, 1972]. We have previouslyproposed an incidence estimate of 1:40,000 [Zerres etal., 1984]. Based on a comparison between our experi-ence with ARPKD and the experience with other dis-eases like cystic fibrosis, Duchenne’s muscular dystro-phy, and spinal muscular atrophy in which the diseaseincidence is well established, we estimate that the in-cidence of ARPKD is about 1:20,000. We therefore sug-gest that genetic counselors use a heterozygosity fre-quency of 1:70 until more precise data are available.Efforts to more precisely define the incidence ofARPKD are in progress.

Diagnostic Criteria/Differential Diagnosis

As long as a direct mutation analysis in ARPKD isnot available, the correct clinical diagnosis in an af-fected child is the most critical point regarding the re-liability of haplotype-based prenatal diagnosis ofARPKD. In families where a pathoanatomical exami-nation of an affected child is available, the diagnosisusually can be made without serious diagnostic doubts.For affected children who survive the perinatal periodwhere no pathoanatomical data are available, the di-agnosis can be difficult, particularly in those caseswithout sonographically detectable liver involvement.However, the radiological criteria regarding the kid-neys [Garel, 1984] cannot be regarded as pathogno-monic for ARPKD, because the renal findings in otherconditions can be sonographically indistinguishable.Results of a prenatal ultrasound examination in a pre-viously affected child are of great value and should al-ways be taken into consideration. In particular, oligo-hydramnios before 20 weeks gestation is unusual inARPKD, as is the presence of discrete cysts in the fetalultrasound.

The most important differential diagnosis of ARPKDis early-onset autosomal dominant polycystic kidneydisease (ADPKD). Therefore, we agree with Ogborn[1994] who stated in an invited review on polycystickidney disease: ‘‘Perhaps the single most useful inves-tigation in the evaluation of a child with early onset ofcystic renal disease is ultrasound of the parents . . .

Thus a negative ultrasound of both parents reduces theprobability of a diagnosis of ADPKD to the level of aspontaneous mutation.’’ (p. 763). At the age of 30, anormal renal ultrasound scan usually rules out AD-PKD [Bear et al., 1992].

Other conditions such as early-onset ADPKD, spo-radic glomerulocystic kidney disease, tuberous sclero-sis, juvenile nephronophthisis, nephroblastomatosis,radiolucent bilateral Wilms tumor, bilateral renal veinthrombosis, radiocontrast nephropathy, and kidney in-volvement in syndromes like Meckel- or Bardet-Biedlsyndrome may mimic ARPKD [Guay-Woodford, 1996].However, these conditions usually can be differenti-ated from ARPKD on the basis of family history as wellas clinical, radiologic, or histopathologic data.

Congenital hepatic fibrosis can be associated withseveral of the renal cystic diseases listed above, includ-ing sporadic glomerulocystic kidney disease, tuberoussclerosis, juvenile nephronophthisis, as well as withMeckel syndrome, Jeune syndrome, different short ribpolydactyly syndromes and Ivemark syndrome [Zerreset al., 1984; Bernstein and Slovis, 1992]. Congenitalhepatic fibrosis has been observed in rare cases ofADPKD as well [Cobben et al., 1990]. A positive familyhistory leads to the definite diagnosis of ADPKD inthese cases. Congenital hepatic fibrosis may also beassociated with other liver lesions such as Caroli’s dis-ease and choledochal cysts [Summerfield et al., 1986].The frequent association of Caroli’s disease withARPKD is well established [Takehara et al., 1989].Therefore, in ARPKD patients, gross cystic dilatationof the intrahepatic biliary tree (Caroli’s disease) shouldbe regarded as a clinical manifestation of the ductalplate malformation.

DNA Analysis/Genetic Map

The ARPKD interval is flanked by several highlypolymorphic markers and therefore, the majority offamilies is informative for haplotype-based analysis(Mucher et al., in press). Longer PCR products (>200bp) often cannot be amplified from DNA extracted fromparaffin-embedded material or microscopic slides;therefore, multiplex PCR often can not be applied inthese cases.

The genetic distance of the closest flanking markersis less than 4 cM (Fig. 1). In cases without evidence fora recombination event between the flanking markers,the probability of a false diagnosis due to a possibledouble recombination is probably much less than 0.1%.Recombination mapping data indicate that the locationof the ARPKD gene is probably much closer to the telo-meric flanking markers than to the proximal markerD6S466. Based on these observations, we concludedthat the three fetuses with recombination events in theARPKD interval probably carried a greater than 50%risk of having ARPKD. In each case, the diagnosis wassubsequently confirmed with histopathologic analysis.

Heterogeneity/Formal Genetics

The issue of possible locus heterogeneity in ARPKDhas been a matter of long-standing debate. Blyth andOckenden [1971] hypothesized, on the basis of the in-

142 Zerres et al.

trafamilial variability of ARPKD in affected siblings,that the four phenotypes are distinct disease entitiescaused most likely by different mutant genes. As analternative formulation we have proposed that ARPKDresults from multiple allelism. This genetic model sug-gests that a few different mutant alleles of a singlegene could account for both the relatively high level ofphenotypic concordance within families as well as thebroad range of phenotypes that is evident among dif-ferent families [Zerres et al., 1984].

The question of genetic heterogeneity is closely re-lated to the definition of diagnostic criteria of ARPKDin at least one previously affected child. On the basis ofstrict pathoanatomic criteria there is no evidence ofgenetic heterogeneity. This observation is in accor-dance with other recent linkage studies including morethan a total of 200 families with the full spectrum ofrenal and hepatic manifestations with a total lod scoreof more than 50 (Mucher, personal communication).These linkage data support the hypothesis that mul-tiple mutant alleles of a single gene are responsible fora broad clinical spectrum of ARPKD.

On the basis of our material, in addition to results oflinkage studies in families fulfilling the mentionedstrict diagnostic criteria of ARPKD, the remaining riskof a second gene locus should not be greater than 1%,which obviously is a rough estimate only. The risk of afalse diagnosis is probably greater in cases where nopathological examination has been performed in an af-fected child.

The remaining low potential risk of a false diagnosisdue to phenocopies should be addressed in the geneticcounseling session.

Fetal Morphology of ARPKD

Necropsy of affected fetuses aborted after prenataldetection by ultrasound can detect evidence of ARPKDat 20–40 weeks gestation [Zerres et al., 1988]. Histo-pathology has shown that the enlargement of the in-terstitial portions of the collecting tubules in ARPKDoccurs in kidneys that have initially developed nor-mally [Potter, 1972]. Consequently, it has been sug-gested that the renal alterations are unlikely to be seenin early fetal development. The timing of the in uterodevelopment of the ductal plate malformation, which isinvariably associated with ARPKD, is unknown. Blythand Ockenden [1971] confirmed the presence of occa-sional cortical and medullary cystic tubules in both kid-neys of twin fetuses with ARPKD who were examined14 weeks after conception, but the liver was not re-tained for histology. We have previously demonstratedthe typical renal and biliary changes in a single caseafter termination at the 14th week of gestation [Zerreset al., 1995]. The fetal histopathological findings re-ported in the current series are the earliest morpho-logic kidney and liver changes documented to date.Moreover, our results clearly demonstrate that congen-ital hepatic fibrosis is invariably associated withARPKD even very early in fetal development.

In all the fetuses with early histopathologic evidenceof ARPKD, previously affected siblings suffered fromperinatal-onset, severe ARPKD. We propose that his-

topathologic changes in the subsequent fetal siblingsrepresent the same severe manifestation. By extension,kidney and liver development early in gestation mayappear to be normal in fetuses predicted to be affectedby DNA analysis, if previously affected siblings havemanifested a milder ARPKD phenotype.

CONCLUSIONS

Our experience with a haplotype-based approach forthe prenatal diagnosis of ARPKD indicated that themethod is both robust and reliable. The vast majority ofthe families were informative with the highly polymor-phic markers of the ARPKD interval and to date, wehave observed precise correlation between the geno-type and the predicted phenotype. Typical kidney andliver manifestations were evident in all affected fetusesas early as the 13th week of gestation. The validity ofthis analysis depends primarily on establishing the di-agnosis of ARPKD in a previous sibling. Current sono-graphic modalities are usually sensitive enough to es-tablish the diagnosis in a surviving child. Histopatho-logic analyses should be reserved for unusual cases. Inaddition, a negative renal sonogram in both parents isan indispensable diagnostic finding.

In our test cohort, we have no evidence for geneticheterogeneity. While our data cannot absolutely ex-clude the possibility of a second ARPKD locus on thebasis of strict diagnostic criteria, the remaining risk isprobably low. A rough estimate of less than 1% can beused for genetic counseling unless more data are avail-able.

ACKNOWLEDGMENTS

The authors thank the families for their cooperation.In addition, we thank Dr. Jay Bernstein and Dr. Cor-nelia Tennstedt for their critical analysis of diagnosti-cally challenging cases. This study was supported bythe Deutsche Forschungsgemeinschaft (KZ) and NIHgrant R01DK51034 (LG-W). We thank Dennis Fiegenfor technical help.

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