Spectrum of Mutations in the Gene for Autosomal Recessive Polycystic

J Am Soc Nephrol 13: 76–89, 2003

Spectrum of Mutations in the Gene for Autosomal Recessive Polycystic Kidney Disease (ARPKD/PKHD1) CARSTEN BERGMANN,* JAN SENDEREK,* BEATE SEDLACEK,* IOANNIS PEGIAZOGLOU,* PATRICIA PUGLIA,* THOMAS EGGERMANN,* ¨ NEBORN,* LASZLO FURU,† LUIZ F. ONUCHIC,‡§ SABINE RUDNIK-SCHO ¶# MONICA DE BACA, GREGORY G. GERMINO,‡ LISA GUAY-WOODFORD,储 ¨ TTNER,¶ and STEFAN SOMLO,† MARKUS MOSER,& REINHARD BU KLAUS ZERRES * *Institute of Human Genetics, Aachen University, Aachen, Germany; †Medicine and Genetics, Yale University, New Haven, Connecticut; ‡Medicine and Genetics, Johns Hopkins University, Baltimore, Maryland; § Medicine, University of Sao Paulo, Sao Paulo, Brazil; ¶Institute of Pathology, University of Bonn, Germany; # Department of Pathology, Thomas Jefferson University, Philadelphia, Pennsylvania; 储Medicine and Pediatrics, University of Alabama at Birmingham, Birmingham, Alabama; &Max-Planck-Institute for Biochemistry, Martinsried, Germany.

Abstract. Autosomal recessive polycystic kidney disease (ARPKD/PKHD1) is an important cause of renal-related and liver-related morbidity and mortality in childhood. Recently mutations in the PKHD1 gene on chromosome 6p21.1-p12 have been identified as the molecular cause of ARPKD. The longest continuous open reading frame (ORF) is encoded by a 67-exon transcript and predicted to yield a 4074 –amino acid protein (“polyductin”) of thus far unknown function. By now, a total of 29 different PKHD1 mutations have been described. This study reports mutation screening in 90 ARPKD patients and identifies mutations in 110 alleles making up a detection rate of 61%. Thirty-four of the detected mutations have not been reported previously. Two underlying mutations in 40 patients and one mutation in 30 cases are disclosed, and no mutation was detected on the remaining chromosomes. Mutations were found to be scattered throughout the gene without evidence of clustering at specific sites. About 45% of the

changes were predicted to truncate the protein. All missense mutations were nonconservative, with the affected amino acid residues found to be conserved in the murine polyductin orthologue. One recurrent missense mutation (T36M) likely represents a mutational hotspot and occurs in a variety of populations. Two founder mutations (R496X and V3471G) make up about 60% of PKHD1 mutations in the Finnish population. Preliminary genotype-phenotype correlations could be established for the type of mutation rather than for the site of the individual mutation. All patients carrying two truncating mutations displayed a severe phenotype with perinatal or neonatal demise. PKHD1 mutation analysis is a powerful tool to establish the molecular cause of ARPKD in a given family. Direct identification of mutations allows an unequivocal diagnosis and accurate genetic counseling even in families displaying diagnostic challenges. [email protected]

Autosomal recessive polycystic kidney disease (ARPKD [MIM 263200]) or polycystic kidney and hepatic disease 1 (PKHD1) is a severe inherited disorder with a proposed incidence of 1/20,000 live births and a widely variable clinical spectrum (1–3). Its principal manifestations involve the fusiform dilation of renal collecting ducts and distal tubuli and dysgenesis of the hepatic portal triad. The only signs potentially displayed in utero, albeit often not even visible in second

trimester fetal sonography, are enlargement and increased echogenicity of both kidneys as well as oligohydramnios (4 – 6). As many as 30 to 50% of affected neonates die shortly after birth in respiratory insufficiency due to pulmonary hypoplasia. In ARPKD patients surviving the neonatal period, the prognosis is more optimistic (7). Frequent complications include systemic hypertension (HTN), end-stage renal disease (ESRD), and clinical manifestations of congenital hepatic fibrosis (CHF). Due to the poor prognosis of early manifestations of ARPKD, there is a strong demand for prenatal diagnosis (6). An early and reliable prenatal diagnostic test became feasible in 1994, when the ARPKD gene was mapped to chromosome 6p21-cen (8). With current knowledge from linkage data, there is no clear evidence of genetic heterogeneity in ARPKD. Two independent groups recently unraveled the PKHD1 gene (9,10). Ward et al. identified the human gene by homology to

Received August 2, 2002. Accepted September 13, 2002. Correspondence to Dr. Carsten Bergmann, Institute of Human Genetics, Aachen University, Pauwelsstra␤e 30, D-52074 Aachen, Germany. Phone: ⫹49-2418080178; Fax: ⫹49-241-8082580; E-mail: [email protected] 1046-6673/1312-0076 Journal of the American Society of Nephrology Copyright © 2002 by the American Society of Nephrology DOI: 10.1097/01.ASN.0000039578.55705.6E


the mutation in the PCK rat model for polycystic kidney disease (11,12), and our group succeeded by positional cloning (13–16). On genomic DNA, the gene spreads over an expanse of at least 470 kb. A minimum of 86 exons is assembled into a variety of alternatively spliced transcripts sized from approximately 8.5 kb to approximately 13 kb (10). The longest continuous open reading frame (ORF; Figure 1) is encoded by a 67-exon transcript and is predicted to yield a protein of 4074 amino acids. The complex pattern of splicing was found to be highly conserved in the murine orthologue (17). The predicted full-length protein that we termed polyductin represents a novel integral membrane protein with an extensive, highly glycosylated N-terminal extracellular region, a single transmembrane-spanning domain, and a short cytoplasmic tail containing potential phosphorylation sites. Polyductin might function as receptor or ligand. The proposed extracellular domain contains several IPT (Ig-like, plexin, transcription factor) domains that are shared by hepatocyte growth factor (HGF) receptor and the plexin superfamily involved in the regulation of cellular adhesion, repulsion, and proliferation (18 –20). In addition, multiple PbH1 (parallel beta-helix 1) repeats are found in the putative polyductin extracellular region. PbH1 repeats are present in virulence factors, adhesins, and toxins in bacterial pathogenesis and known to bind to carbohydrate moieties (21). In case of polyductin, targets for binding could include glycoproteins on the cell surface or in the basement membrane. However, definite polyductin functions remain to be unraveled. By now, a total of 29 different PKHD1 mutations have been described. The present study aimed at three major points: (1) to define the underlying molecular basis in a cohort of 90 apparently unrelated families with at least one child affected by ARPKD; (2) to determine the achievable mutation detection rate in PKHD1; and (3) to evolve possible genotype-phenotype correlations.

Spectrum of PKHD1 Mutations

77

for PKHD1 mutation screening. This cohort mainly consisted of families who had sought prenatal diagnosis during the last decade. The diagnostic criteria were the same as those reported elsewhere (6): (1) clinical manifestation of ARPKD with characteristic ultrasonographic findings (22,23) and (2) presence of at least one of the following: (a) absence of renal cysts in parental ultrasound (this criterion was fulfilled in all but three families), (b) symptoms or histopathologic evidence of hepatic fibrosis, (c) pathoanatomical proof of ARPKD in an affected sibling, or (d) parental consanguinity. The cohort of patients studied represented diverse nationalities from 24 mainly European countries and the entire clinical spectrum (Tables 1 through 3). Phenotypes were categorized as severe or moderate. The group of severe cases included 44 families with perinatal or neonatal demise of affected children (Table 1). The moderate cohort comprised 38 families with patients who either survived complications (mainly respiratory) during the first month of life or became first symptomatic beyond the neonatal period (Table 3). We are aware of the difficulties in establishing phenotype categorization, particularly with regard to the group of patients denoted as moderately affected. A further subdivision of this cohort was hardly feasible due to diversity of clinical presentation. Eight families were characterized by marked intrafamilial phenotypic variability among affected siblings (Table 2). DNA from the 90 ARPKD families studied and 150 apparently unrelated, healthy control individuals was obtained after informed consent had been given.

Haplotype Analysis Families in which a prenatal diagnosis had been established were compatible with linkage to the ARPKD locus on chromosome 6p. At least four informative markers from a set of 15 microsatellites from the ARPKD interval had been typed in each family (10,24). After identification of a recurrent PKHD1 mutation, allele-sharing analysis was done in the respective families to study a possible founder effect. Primers for PCR amplification were as published by the Genome Data Base (http:// www.gdb.org) and by Mu¨ cher et al. (24) and Onuchic et al. (16). Sense primers were labeled with FAM fluorophores (Pharmacia, Uppsala, Sweden) for electrophoresis and analysis on an ABI PRISM 377 genetic analyzer (Applied Biosystems, Weiterstadt, Germany).

Materials and Methods Selection of the Study Population

Mutation Analysis

A series of 90 apparently unrelated families with at least one child affected by ARPKD (148 affected individuals/fetuses) was selected

Mutation screening was done for the 67 exons that constitute the longest continuous polyductin ORF. Genomic DNA from an affected

Figure 1. Linear representation of the polyductin protein encoded by the longest potential open reading frame. The sites of mutations detected in this study and described in the literature are indicated. Putative polyductin domains are colored (red, signal peptide; yellow, IPT; green, PbH1 repeat; blue, transmembrane domain). The lengths of truncated peptides encoded by PKHD1 alleles with chain-terminating mutations are illustrated at the bottom.

78

Journal of the American Society of Nephrology


Table 1. Synopsis of ARPKD patients/families with severe phenotype Familya 215 (C)

Turkey

228

Finland

251 (C)

Turkey

290 (C) 330 (C) 340 385 413 (C) 440 (C) 470 (C) 483 108 118 133

Genotypeb

Origin Ex33: Ex33: Ex16: Ex16:

D1793fsX1802 (P) D1793fsX1802 (M) R496X (P) R496X (M)

Ex14: P356fsX357 (P) Ex14: P356fsX357 (M) Turkey Ex14: P356fsX357 (P) Ex14: P356fsX357 (M) Finland Ex16: R496X (P) Ex16: R496X (M) United Kingdom Ex11: S237fsX244 (P) Ex36: L1966fsX1969 (M) Finland Ex16: R496X (P) Ex16: R496X (M) Inuit (Canada) Ex32: G1320fsX1322 (P) Ex32: G1320fsX1322 (M) Turkey Ex61: 13658fsX3664 (P) Ex61: I3658fsX3664 (M) Syria Ex58: R3107X (P) Ex58: R3107X (M) Finland Ex16: R496X (P) Ex16: R496X (M) Finland Ex16: R496X (P) Ex61: V3471G (M) Germany Ex3: T36M (P) Ex3: F42fsX63 (M) Finland Ex16: R496X (M) Ex61: V3471G (P)

165

Finland

Ex16: R496X (P) Ex61: V3471G (M)

219

Finland

353

United States

95 (C)

Turkey

Ex16: R496X (P) Ex52: D2761Y (M) Ex3: T36M (M) Ex16: G470fsX480 (P) Ex16: R496P (P) Ex16: R496P (M)

Patient Gender

Deathc

Additional Findingsd

215.1

m

Perinatal

OH, enlarged kidneys (30gw), RD, PM

228.1 228.2 228.3 251.1 251.2 290.1 290.2 330.1

m f f f f f m f

Perinatal CVS/TOP CVS/TOP Perinatal US/TOP Perinatal CVS/TOP Perinatal

OH, RD, PM PM

340.1

m

Perinatal

OH (30gw), RD, PM

385.1

m

Perinatal

RD, PM

413.1

f

Perinatal

OH, enlarged kidneys, PSP (26gw), RD, PM

440.1

m

Perinatal

OH (24gw), RD, PM

470.1

m

Perinatal


483.1

f

Perinatal

OH (20gw), RD, PM

108.1 108.2 118.1

f m f

Perinatal US/TOP Perinatal

RD, PM OH, PSP (22gw), PM OH, enlarged kidneys, PSP (32gw), RD, PM

133.1 133.2 133.3 165.1 165.2 165.3 165.4 219.1

f m f m m f m m

Perinatal US/TOP CVS/TOP Perinatal CVS/TOP CVS/TOP CVS/TOP Perinatal

RD, PM OH, enlarged kidneys, PSP (22gw), PM PM OH, RD, PM PM

353.1

m

Perinatal

OH (34gw), RD, PM

95.1 95.2 95.3 95.4 95.5

m f f m f

Perinatal Perinatal Perinatal CVS/TOP CVS/TOP

RD RD RD, PM PM

individual of each family was amplified by PCR with oligonucleotide primers complementary to flanking intronic sequences. Primers were designed using the Primer3 program (http://www-genome.wi.mit.edu/ cgi-bin/primer/primer3.cgi) to generate PCR products of less than 300 bp suitable for single-strand conformation polymorphism analysis (SSCP). Larger exons were split into up to ten overlapping fragments of reasonable size for SSCP. In total, PKHD1 was amplified as 90 fragments. For SSCP screening, PCR products were run at four different conditions on 10% non-denaturing polyacrylamide gels (49:1 acrylamide:bis-acryl-

OH, RD, PM OH, enlarged kidneys, PSP (29gw), PM OH, enlarged kidneys, PSP (34gw), RD, PM PM OH (32gw), RD, PM

OH, RD, PM

amide with or without 10% glycerol) at 250 V for 2.5 to 4 h either at room temperature or 4°C. For visualization of bands, gels were silver stained. Samples exhibiting aberrant migration patterns on SSCP gels were subjected to direct sequence analysis. PCR products were gel purified with the QiaEx-kit (Qiagen, Hilden, Germany) and sequenced employing ABI BigDye chemistry (Applied Biosystems, Weiterstadt, Germany). The same primers as for SSCP were used as sequencing primers. Samples were run and analyzed on an ABI PRISM 310 genetic analyzer (Applied Biosystems). When a mutation had been identified, segregation of the



79

Table 1. Continued Familya

Origin

Genotypeb

Patient

Gender

111

Germany

188 (C)

Pakistan

Ex3: T36M (M) Ex27: M997K (P) Ex9: G223S (P) Ex9: G223S (M)

225 (C)

Netherlands

Ex21: D703N (P) Ex21: D703N (M)

229

Finland

297 (C)

Israel Arab

Ex3: T36M (M) Ex57: I2957T (P) Ex61: R3482C (P) Ex61: R3482C (M)

111.1 111.2 188.1 188.2 188.3 188.4 188.5 225.1 225.2 225.3 229.1

360 (C)

Israel Arab

Ex61: R3482C (P) Ex61: R3482C (M)

197

Germany

451

Finland

264 (C)

Turkey

427

United Kingdom

90

France

91

France

Ex3: T36M (P) Ex61: L3413fsX3432 Ex16: R496X (P) Ex16: R496X (M) Ex14: R328X (P) Ex14: R328X (M) Ex3: T36M (M) Ex61: L3494fsX3528 Ex36: L1966fsX1969 Ex39: L2134P (P) [Ex58: I3081V (P)] Ex36: L1966fsX1969

182 200

Finland Denmark

Ex16: R496X (P) Ex7: T166fsX178 (M)

(M)

(P) (M)

(P)

Deathc


f m m m f m f f f m f

Perinatal Perinatal Perinatal Perinatal Perinatal Perinatal Perinatal Perinatal Perinatal CVS/TOP Perinatal

RD, PM RD, PM OH, RD, PM RD RD RD RD OH, enlarged kidneys, PSP (34gw), RD, PM OH, enlarged kidneys, PSP (31gw), RD, PM

297.1 297.2 297.3 360.1 360.2 360.3 197.1 197.2 451.1

f m m m f f m m m

Perinatal Perinatal CVS/TOP Perinatal Perinatal Perinatal 1w 1m 2w

RD OH (28gw), RD, PM RD RD RD, PM OH, RD, HTN, PM RD, SC 0.8, HTN, anemia, septicemia, PM RD, HTN, SC 4.6 (2w)

264.1

f

3w

OH, RD, HTN, ESRD, septicemia, PM

427.1

m

3w

OH, RD, HTN, anuria, SC 5.2 (3w), PM

90.1

f

1m

SC 0.5, HTN, enlarged liver, portal HTN, liver failure due to ascending cholangitis, PM

91.1 91.2 182.1 200.1 200.2

m f f m m

Perinatal CVS/TOP Perinatal Perinatal Perinatal

RD, PM

altered allele was tested by direct sequencing of parental probes. DNA samples from 150 apparently unrelated normal control subjects were also tested under appropriate SSCP conditions to exclude a possible polymorphism in case of a potential missense mutation or subtle in-frame change.

Results Mutation Analysis in PKHD1 In a cohort of 90 apparently unrelated families with at least one child affected by ARPKD, we performed a systematic mutation screen of all 67 exons comprised in the longest continuous polyductin ORF. A total of 110 out of expected 180 mutations were observed, resulting in a detection rate of 61%. We were able to disclose two underlying mutations in 40 individuals (45%, for examples see Figure 2) and one mutation in 30 cases (33%). No mutation could be found in 20 patients (22%). In all individuals found to harbor two mutations, the

RD, PM

OH, RD, PM RD, PM OH, enlarged kidneys (31gw), RD

mutant alleles were proven to reside on separate chromosomes (Tables 1 through 3). Of the observed changes, 49 were chain-terminating mutations comprising 26 nonsense mutations, 21 deletions/insertions, and 2 splice-site variants. Conclusively, about 45% of the mutations observed are predicted to truncate the protein. A second group of probable mutations were non-conservative missense changes for which segregation with the disease was proven. These alterations (total 61) were neither found on control chromosomes nor previously described as polymorphisms (9,10). All observed missense changes were found to replace residues conserved in the murine polyductin orthologue (Table 4). All probands with putative missense substitutions were included in the screen of the rest of the gene. No more than a maximum of two putative pathogenic mutations were observed in all but one case. In this patient (90.1), we identified

80



Table 1. Continued Familya

Origin

Genotypeb

Patient

Gender

254 282

United Kingdom Australia

Ex24: P805L (P) Ex3: T36M

333 337 384 452

Finland United Kingdom Germany Germany

Ex16: R496X (M) Ex3: T36M Ex61: R3482C (P) Ex3: T36M

494 126 185

Finland Italy Germany

Ex16: R496X (P)

220

Albania

226

Germany

441

Germany

254.1 282.1 282.2 282.3 333.1 337.1 384.1 452.1 452.2 494.1 126.1 185.1 185.2 220.1 220.2 220.3 226.1 226.2 441.1

f f f m m m f m m m m m m m f m m f f

Deathc


Perinatal Perinatal CVS/TOP CVS/TOP Perinatal Perinatal Perinatal Perinatal US/TOP Perinatal Perinatal Perinatal Perinatal Perinatal US/TOP US/TOP Perinatal CVS/TOP Perinatal

OH, RD, PM OH, RD, PM PM OH, RD, PM RD, PM OH, RD, PM RD, PM OH, enlarged kidneys, OH (35gw), RD, PM OH, enlarged kidneys, OH, enlarged kidneys, OH, enlarged kidneys, OH, enlarged kidneys, OH, enlarged kidneys, OH, enlarged kidneys, OH, enlarged kidneys, PM OH (33gw), RD, PM

PSP (22gw), PM PSP PSP PSP PSP PSP PSP PSP

(34gw), (36gw), (28gw), (31gw), (22gw) (23gw), (31gw),

RD, RD, RD, RD,

PM PM PM PM

PM RD, PM

a

(C), parental consanguinity known. (P), paternally inherited allele; (M), maternally inherited allele. c CVS/TOP, prenatal diagnosis after chorionic villus sampling (CVS) based on haplotyping with subsequent termination of pregnancy (TOP); US/TOP, ultrasound findings suspicious of ARPKD with subsequent termination of pregnancy (TOP); †, deceased; w, weeks; m, months; y, years; RTX, kidney transplantation. d OH, oligohydramnios; PSP, “pepper and salt” pattern in prenatal US; gw, gestational week; RD, respiratory distress; PM, postmortem with characteristic ARPKD findings in liver and kidneys; CLKT, combined liver-kidney transplantation; SC, serum creatinine (mg/dl); ESRD, end-stage renal disease; portal HTN, portal hypertension; HTN, arterial hypertension. b

two missense changes residing on the paternal chromosome that were not detected in control individuals. However, one of these alterations was a conservative exchange from isoleucine to valine, making its pathogenic character questionable. The mutations we detected were mostly unique and scattered throughout the entire gene. We observed one recurrent missense mutation in exon 3 changing the ACG codon 36 to ATG (c.107C⬎T; T36M). This alteration occurred in 26 obviously unrelated patients of different ethnic origin. Allele sharing analyses proved that at least 16 different haplotypes are responsible for the high prevalence of this mutation in the German and Finnish populations (data not shown). Interestingly, individuals 2.1 and 2.2 of family 2 harboring T36M in the homozygous state carry different parental haplotypes (Figure 3). As previously suggested (24), two founder alleles are responsible for about 60% of PKHD1 mutations in the Finnish cohort. We identified the PKHD1 mutations associated with these two consensus haplotypes (c.1486C⬎T [R496X] and c.10412T⬎G [V3471G]). Table 4 summarizes all pathogenic changes that were either predicted to truncate the protein or represented missense mutations not detected in the control population. Figure 1 indicates the relative location of each mutation along a linear representation of the protein. In addition to the reported mutations, several polymor-

phisms were observed. Some of these have been described previously (9). Overall, SSCP detected 26 recurrent (up to 40% of chromosomes) and 7 unique aberrant migration patterns that were identified as polymorphisms because they were (1) intronic changes beyond the consensus splice sites, (2) located in the 5' or 3' UTR, (3) silent exonic changes (not altering an amino acid), or (4) a substitution that did not segregate with the disease or was found in normal controls. Two novel missense changes (c.1736C⬎T [T579M] and c.2489A⬎G [N830S]) could be excluded as pathogenic mutations as they were also detected in about 10% of normal control individuals.

Genotype-Phenotype Correlations In the cohort of patients with a moderate phenotype, we were able to detect 30 out of 76 mutations (40%), whereas we identified 68 mutations in the 88 ARPKD alleles (77%) from individuals displaying a severe phenotype. Conclusively, the level of detection was considerably higher in patients with a severe clinical course. The detection rate observed in the group of families with phenotypic variability among affected sibs reached 75% (12 out of 16 alleles); however, these latter figures might be biased due to the small number of families screened in this cohort. Analysis of the mutational spectrum in the different subgroups revealed more than half of the mutations detected in the severe

f m f m m f

3.1 3.2 127.1 127.2 316.1 316.2

m f m

151.2 1.1 1.2

m f f

59.3 2.1 2.2 f m f m

m

59.2

105.1 105.2 105.3 151.1

m

59.1

Renal Involvementd

Liver Involvemente

† (perinatal) † (perinatal) 7y SC 0.3 (1y), SC 0.7 (7y) 6y SC 0.6, Clcrea 27 (1y), SC 1.0 (6y) † (perinatal) † (1m) 15y SC 0.6, Clcrea 61 (5y), SC 2.2 (15y) 18y SC 0.4, Clcrea 131 (2y), SC 2.8 (18y) † (1m) ESRD (1m) 6y ESRD (2y), RTX (2y), SC 0.9 (6y) † (perinatal) † (perinatal) 24y ESRD, RTX (9y), rejection (11y), RTX (15y), rejection (22y)

† (2y) SC 2.1 (1y), ESRD (2y) † (perinatal) 5y SC 3.0 (3y), ESRD (5y)

† (1y)

bx CHF, Caroli syndrome (23y)

Enlarged

bx CHF (2y)

bx CHF (2y)

US n (7y)

echog.1 (5y)

echog.1 (1y)

SC 1.6, Clcrea 15 (neonatal), echog. 1 (4m) SC 2.9 (10m) † (perinatal) SC 0.8 (birth)

Age at Last Patient Gender Examination/ Deathc Additional Findingsg

RD, PM RD neonatally, anemia, growth retardation RD, PM RD, PM

Yes (2y)

RD, PM RD, PM Anemia, awaiting CLKT (24y)

Yes (birth) RD neonatally, PM Yes (birth)

Yes (5y)

Yes (2y)

OH, RD, PM RD, PM RD (first 4 days)

Yes (1y) Yes (birth) OH

Yes (2y)

Yes (birth)


Yes (birth) OH, anemia

HTNf

b

(C), parental consanguinity known. (P), paternally inherited allele; (M), maternally inherited allele. c CVS/TOP, prenatal diagnosis after chorionic villus sampling (CVS) based on haplotyping with subsequent termination of pregnancy (TOP); US/TOP, ultrasound findings suspicious of ARPKD with subsequent termination of pregnancy (TOP); †deceased; w, weeks; m, months; y, years. d Renal ultrasound findings were always typical of ARPKD as an obligate inclusion criterion. SC, serum creatinine (mg/dl); Clcrea, creatinine clearance (ml/min per 1.73 m2); ESRD, end-stage renal disease; RTX, kidney transplantation. e US, ultrasound; MRI, magnetic resonance imaging; bx, liver biopsy; CHF, congenital hepatic fibrosis; echog. 1, increased liver echogenicity; portal HTN, portal hypertension; HSM, hepatosplenomegaly; LTX, liver transplantation. f HTN, arterial hypertension. g OH, oligohydramnios; PSP, “pepper and salt” pattern in prenatal US; gw, gestational week; RD, respiratory distress; PM, post mortem with characteristic ARPKD findings in liver and kidneys; CLKT, combined liver-kidney transplantation.

a

Germany Ex 61: R3482C

316

IVS13-1G⬎A (P) Ex43: I2331K (M)

United Ex3: T36M Kingdom

Finland

3

Ex3: T36M (P) Ex61: V3471G (M)

127

Finland

1

Germany Ex21: W656C (M) Ex30: G1123S (P)

151

Ex9: I222V (M) Ex58: R3240X (P)

Ex3: T36M (P) Ex3: T36M (M)

Italy

Finland

2

Ex58: R3107X (P) Ex59: D3293V (M)

Genotypeb

105

Turkey

Origin

59

Familya

Table 2. Synopsis of ARPKD families with intrafamilial phenotypic variability

J Am Soc Nephrol 13: 76–89, 2003 Spectrum of PKHD1 Mutations 81

Greece

Czech Republic

Germany

457

277

357

Slovenia

Germany

324

231

Germany

483

Germany

Germany

27

9

Germany

106

Germany

Portugal

77

195

Israel-Arab

57

Germany

Finland

299

41

Finland

56

Germany

Germany

80

Germany

Finland

113

485

Turkey

270 (C)

163

Turkey

Origin

260 (C)

Familya

Ex3: T36M

Ex3: T36M

Ex3: T36M

m f

231.2

m

231.1

f

9.2

m

f

f

f

f

9.1

195.1

41.1

163.1

485.1

357.1

f

f

277.1

f

457.2

m

457.1

324.1

f

f

27.2 483.1

f

27.1

m

f

77.2 106.1

m

m

f

f

77.1

57.1

299.1

56.1

m

f

f

m

f

Gender

18y

21y

8y

18y

14y

12y

7y

4y

4y

4y

US/TOP

4y

3y

1y

† (3y)

6y

11y

CVS/TOP

12y

2y

† (2y)

17y

8y

4y

5y

2y

1y

Age at Last Examination/Deathc

Yes (1y)

HSM, echog. 1, dilated biliary

Yes (birth) biliary ducts (7y)

Clcrea 43 (7y)

SC 3.3 (18y)

ESRD, dialysis (14y), SC 8.5 (21y)

SC 0.7 (2y), 0.6 (8y)

SC 0.5 (12y), 0.7 (18y)

SC 0.6 (14y)

SC 1.3, Clcrea 64 (6y), SC 1.7, Clcrea 45 (12y)

portal HTN (10y)

HSM (1y), echog.1,

portal HTN (12y)

HSM (2y), echog.1,

echog.1 (8y)

US n (18y)

US n (14y)

Yes (3y)

Yes (2y)

No (8y)

No (18y)

No (14y)

bx CHF (3y), HSM, portal HTN (11y) Yes (2y)

Yes (1y)

SC 0.5, Clcrea 105 (4y)

bx CHF, dilated biliary ducts (3y)

Yes (birth)

Yes (2m)

US n (1y), HSM, echog.1, dilated

ducts (5m)

MRI: CHF, ascites, dilated biliary

HSM, echog.1 (2m)

Yes (birth)

Yes (6m)

echog.1 (1y) US n

Yes (birth)

Yes (birth)

No (6y)

Yes (1y)

Yes (1y)

Yes (birth)

Yes (birth)

echog.1 (1y)

bx CHF (1 1/2y)

bx CHF(2y), enlarged

portal HTN (10y)

bx CHF (1y), portal HTN (2y)

bx CHF (2y)

US n

bx CHF(4y), HSM, portal HTN (14y) Yes (birth)

ducts (8y)

Yes (5y) No (4y)

US n

Yes (birth)

Yes (birth)

HTNf

echog. 1 (4y)

US n (4m), echog. 1 (2y)

US n

Liver Involvemente

SC 0.7, Clcrea 31 (1m), SC 0.3 (1y), 0.7 (3y), 1.2,

SC 0.5 (5m), 1.4 (4y)

SC 1.2 (2m), 0.8 (3y), 1.0, Clcrea 38 (4y)

SC 0.5 (1y), 1.7 (4y)

SC 1.3, Clcrea 38 (3y)

SC 1.0, Clcrea 43 (1y),

SC 0.3 (1y)

SC 2.2 (birth), ESRD, dialysis (1y), SC 5.2 (3y)

SC 4.8 (6y)

SC 0.7 (1y), 3.5 (3y), ESRD, dialysis (4y),

rejection (10y)

SC 1.8 (1y), 3.2 (4y), ESRD (7y), RTX (8y),

SC 1.4 (3y), ESRD (7y), RTX (8y), SC 3.1 (12y)

SC 0.9 (2y)

SC 0.7 (2y)

SC 6.5 (14y), RTX (17y)

SC 1.2 (8y)

SC 0.3, Clcrea 110 (2y),

SC 0.5 (4y)

SC 0.4, Clcrea 110 (5y)

SC 0.5 (2y)

SC 0.4 (1m), 0.6, Clcrea 64 (4m),

SC 0.5 (1y)

Renal Involvementd

thrombocytopenia (12y)

Oesophageal varices,

hyperparathyroidism

Growth retardation, Enuresis, anemia,

Hyperparathyroidism

Thrombocytopenia, anemia

Anemia

Anemia

PM

OH, enlarged kidneys, PSP (19gw),

OH, enlarged kidneys, PSP (28gw)

Anemia (6m)

Caput medusae (1 1/2y), PM

CLKT (11y)

Oesophageal varices (10y), awaiting

varices

Growth retardation, oesophageal

defect

OH, demise due to congenital heart

Oesophageal variceal bleeding (14y)

Growth retardation, anemia

OH (31gw), RD (first w), anemia

Additional Findingsg


Ex3: T36M (P)

Ex3: T36M

Ex3: T36M

Ex3: T36M

Ex3: T36M (M)

Ex3: T36M (M)

Ex3: T36M

Ex3: T36M

Ex3: T36M

Ex32: P1486L (M)

Ex14: G369fsX412 (M)

IVS16⫹1G⬎A

Ex16: R496X (P)

Ex61: V3471G (M)

Ex16: R496X (P)

Ex58:L3116fsX3161(M)

80.1

113.2

Ex27: A1030E (P)

113.1

Ex43: I2331K (M)

270.1

260.1

Patient

Ex16: R496X (P)

Ex37: I1998T (M)

Ex37: I1998T (P)

Ex16: I473S (M)

Ex16: I473S (P)

Genotypeb

Table 3. Synopsis of ARPKD patients/families with moderate phenotype

82 J Am Soc Nephrol 13: 76–89, 2003

Germany

Germany

Germany

Belgium

Israel Arab Germany

Italy Sweden Italy Brazil France

Sweden

Germany

Switzerland Venezuela

Italy

12

43

45

48

53 (C) 73

78 81 84 88 96

97

101

124 135

408

Ex3: T36M

Ex3: T36M

Genotypeb

f

124.1 135.1 135.2 135.3 408.1

101.1

78.1 81.1 84.1 88.1 96.1 96.2 97.1

m m f m f

m

m m m f f f m

f f

48.2

53.1 73.1

m

m

f

48.1

45.1

43.1

f f

f

32.2

12.1 12.2

m

Gender

32.1

Patient

13y 14y 11y 8y 1y

20y

7y 9y 11y 11y 8y 3y 10y

18y 2y 14y

26y

13y

13y

33y 31y

16y

22y

Age at Last Examination/ Deathc

Yes (1y)

HSM, portal HTN (8y), bx CHF (11y) echog.1, HSM, portal HTN (7y) Enlarged, bx CHF (3y), portal HTN (6y) US n (11y) Enlarged (3y), echog.1, portal HTN (11y) Enlarged, echog.1 (8y) Enlarged, echog. 1 (3y) bx CHF (4y), HSM, portal HTN (7y)

1.2, Clcrea 45 (14y) SC 1.1 (7y) ESRD, RTX (4y), SC 0.8 (9y) SC 3.5 (11y) SC 2.5 (3y), ESRD (10y), RTX (11y) SC 1.2 (8y) SC 0.7 (3y) ESRD, RTX (5y), SC 2.1 (10y)

SC SC SC SC SC

0.4 0.9 0.6 0.7 0.8

(2y), 0.9 (13y) (14y) (11y) (8y) (1y)

ESRD, dialysis (16y), CLKT (18y)

Yes (1y) Yes (1y)

Portal HTN, bx CHF (10y)

Enlarged, portal HTN, bx CHF (6y) Enlarged, echog.1, gamma-GT 1, dilated biliary ducts (1y)

echog.1, HSM, portal HTN (5y), bx CHF (16y) US n (13y)

bx CHF (1y)

Yes (1y) No No No Yes (birth)

Yes (5y) Yes (3y) Yes (3y) Yes (birth) Yes (1y) Yes (birth) Yes (birth), normal since RTX Yes (3y)

Yes (3y)

Yes (1y)

Yes (1y)

bx CHF (8y) echog.1 (1y), bx CHF (8y), HSM, portal HTN (13y) HSM, echog.1 (26y)

No (33y) No (31y)

Yes (2y)

Yes (3y)

HTNf

bx CHF (25y) US n (14y)

echog.1, portal HTN, HSM (12y), LTX (22y) echog.1, HSM, portal HTN (8y)

Liver Involvemente

SC 1.4 (1y), 0.7 (4y), SC 1.1, Clcrea 80 (13y), ESRD, dialysis (15y), RTX (19y), SC 2.5 (26y) ESRD, CLKT (11y), SC 2.7 (18y) SC 2.4 (2y) SC 0.6 (1y), 1.0, Clcrea 53 (7y), SC

SC 0.4 (3y), 4.4 (15y), ESRD (16y), RTX (18y), dialysis (22y) SC 0.5 (2y), 1.7 (6y), 5.2 (9y), ESRD (10y), RTX (11y), SC 3.0 (16y) SC 2.3 (33y) SC 0.6 (14y), 1.3 (18y), 1.6 (21y), ESRD (28y), RTX (29y), SC 1.4 (31y) SC 0.8 (4y), SC 3.6, Clcrea 25 (13y)

Renal Involvementd

Oesophageal varices (8y)

Oesophageal variceal bleeding (5y), pancytopenia (17y)


Thrombocytopenia, oesophageal variceal bleeding (8y) Oesophageal varices (6y) Oesophageal varices (7y)


Thrombocytopenia (26y)

Growth retardation, enuresis, anemia, hyperparathyreoidism Growth retardation

Intracranial aneurysms, uncomplicated pregnancy (31y)



Additional Findingsg

b

(C), parental consanguinity known. (P), paternally inherited allele; (M), maternally inherited allele. c CVS/TOP, prenatal diagnosis after chorionic villus sampling (CVS) based on haplotyping with subsequent termination of pregnancy (TOP); US/TOP, ultrasound findings suspicious of ARPKD with subsequent termination of pregnancy (TOP); †, deceased; w, weeks; m, months; y, years. d Renal ultrasound findings were always typical of ARPKD as an obligate inclusion criterion. SC, serum creatinine (mg/dl); Clcrea, creatinine clearance (ml/min per 1.73m2); ESRD, end-stage renal disease; RTX, kidney transplantation. e US, ultrasound; MRI, magnetic resonance imaging; bx, liver biopsy; CHF, congenital hepatic fibrosis; echog. 1, increased liver echogenicity; portal HTN, portal hypertension; HSM, hepatosplenomegaly; LTX, liver transplantation. f HTN, arterial hypertension. g OH, oligohydramnios; PSP, “pepper and salt” pattern in prenatal US; gw, gestational week; RD, respiratory distress; PM, post mortem with characteristic ARPKD findings in liver and kidneys; CLKT, combined liver-kidney transplantation.

a

Germany

Origin

32

Familya

Table 3. Continued

J Am Soc Nephrol 13: 76–89, 2003 Spectrum of PKHD1 Mutations 83

84



Figure 2. PKHD1 mutations in families studied. Representative pedigrees are given on the left, and the mutations identified in the respective families are shown on the right. Sense strand electropherograms from the affected patients are illustrated at the top, and a limited reading frame is given below. In case of a homozygous mutation, the wild-type sequence is depicted in a separate electropherogram.

group to be truncating (40 of 68). In the moderate cohort, missense changes were more than three times as frequent as chain-terminating alterations (22 of 30). Further insight into genotype-phenotype correlations should come from patients homozygous for a given mutation. All individuals carrying a termination-type mutation in the homozygous state died shortly after birth. Missense mutation

R3482C was seen homozygously in two consanguineous Israel-Arab families (297 and 360) with perinatal demise in all five affected children. In three other consanguineous multiplex families (95, 188, and 225), each harboring a different missense mutation, all affected individuals died shortly after birth. In two further inbred families with single affected children carrying a homozygous missense mutation (families 260 and


270), a moderate phenotype was observed. Affected individuals of family 2 with the T36M mutation in the homozygous state displayed intrafamilial phenotypic variability. Given that termination-type mutations all have a uniform effect, one would expect the type and position of the missense change to determine the clinical course in compound heterozygotes with one truncating and one missense mutation in trans. Our preliminary results indicate that missense mutations L2134P and D2761Y are associated with perinatal/neonatal demise. Association with a moderate phenotype was observed for missense mutation A1030E while the phenotype varied in patients with I222V and V3471G. However, except for A1030E, which is located in one of the predicted IPT domains, all affected amino acids reside in regions without apparent homology to known protein sequences. Thus, we could not establish any structure-function relationship for these missense mutations.

Discussion The pathways involved in renal cyst formation and biliary dysgenesis in ARPKD are still widely unknown. As a vital step in unraveling the underlying molecular pathomechanisms in this disorder, the responsible gene was identified by two independent groups (9,10).

Novel and Known PKHD1 Mutations Further insight into the underlying disease mechanisms in ARPKD will be provided by both functional investigations and identification of mutations, which will help to unravel protein regions crucial for proper polyductin structure and function. In this study, we performed SSCP analysis on 90 patients diagnosed with ARPKD for PKHD1 mutations and identified 110 mutations. Conclusively, the level of detection reached 61% of disease chromosomes, comparable with detection rates achieved in the initial screen for other large multiexon genes (25,26). Among the mutations identified, 34 were not reported previously, expanding the spectrum of known PKHD1 mutations from 29 to 63 (Table 4). Mutations were dispersed over the entire gene with no evidence of clustering at specific sites. Most of the mutations detected are of unique character, which is in agreement with the existence of many diverse haplotypes on ARPKD chromosomes (6,8). Of 63 different mutations reported so far, only ten were found in two or more pedigrees from the present study or in the cohorts of others (9,10). The recurrent T36M mutation in exon 3 most probably represents a mutational hotspot. This assumption seems reasonable since most of the CpG dinucleotides are hypermutable, as the cytosine is often methylated and susceptible to spontaneous deamination to form a thymidine (27). Two other recurrent mutations (R496X and V3471G) were restricted to the Finnish isolate. At present, with the exception of T36M in exon 3, we have no clear proof of protein regions that should be screened initially for PKHD1 mutations in “non-isolate” populations. In patients in whom only one or no mutation was found, it is likely that limitations of SSCP technique hampered detection of the second or any variant. Alternatively, the remaining


85

sequence variations may be located in regulatory elements or in additional exons not included in the longest ORF. These sequences have not yet been screened. Moreover, other mutation mechanisms, e.g., gross deletions or genomic rearrangements are not detectable by SSCP. In most cases, however, gross deletions seem less likely, because we observed heterozygosity for various nonpathogenic single-nucleotide changes scattered throughout the PKHD1 gene. Nevertheless, deletions of single or some exons or genomic rearrangements cannot be ruled out with certainty by the methods applied. Among the 20 patients analyzed without any detectable mutation, locus heterogeneity has to be considered, though our own linkage data (6,8,28) and those of others (29) argue against the existence of a second ARPKD gene. Alternatively, in some of these patients the assumed diagnosis of ARPKD might be incorrect. It is noteworthy that most individuals of this cohort displayed a moderate phenotype without pathoanatomical proof of ARPKD. Whether the ratio observed in our study between chainterminating mutations (n ⫽ 49) and missense changes (n ⫽ 61) is the correct representation of PKHD1 alterations remains to be clarified. It has to be noted that one of the founder alleles in the Finnish population (R496X) accounts for a considerable proportion of the identified truncating mutations (20 out of 49). Moreover, it is conceivable that SSCP analysis will more likely detect frameshifting mutations than subtle nucleotide substitutions. Any of the missense changes (and, of course, some of the terminating changes as well) may have untoward effects on splicing not assayed by our methods. For example, in family 188, it might be more plausible to assume an intron 9 donor splice site error than an amino acid exchange from glycine to serine. Whether this hypothesis is correct needs to be established on transcript level, but RNA was not accessible. On the other hand, despite evidence from segregation analysis, screen of normal individuals, lack of further changes in the rest of the gene, and the non-conservative nature of the amino acid substitutions, it cannot be ruled out that some of the PKHD1 missense changes reported so far will ultimately be revealed as nonpathogenic alterations.

Genotype-Phenotype Correlations Multiple allelism and the high rate of compound heterozygotes make it difficult to establish convincing genotype-phenotype relationships in ARPKD. Preliminary correlations could be established for the type of mutation. The proportion of chain-terminating mutations was significantly higher in the severe group (59% of identified alleles) than in the moderately affected cohort (23% of identified alleles). Given the considerable number of Finnish patients in this study, it might be argued that the increased prevalence of termination-type mutations in the severe cohort is simply a result of the ethnic background rather than a true relationship to the phenotype. However, even after exclusion of all Finnish patients the ratio remains comparable (51% versus 16%). In this study, all individuals found to harbor two chainterminating mutations in trans (12 families, Table 1) died shortly after birth, irrespective of the site of the premature stop of translation. In family 440, we observed a homozygous

86



Table 4. Spectrum of PKHD1 mutationsa Type of Mutation Location Nonsense

Deletion/insertion

Splicing

cDNA Change

aa Change

Ex14 Ex16 Ex50 Ex58 Ex58 Ex61 Ex65 Ex3 Ex7 Ex11 Ex14 Ex14 Ex16 Ex18 Ex18 Ex29 Ex32 Ex32 Ex33 Ex36

c.982C⬎T c.1486C⬎T c.8011C⬎T c.9319C⬎T c.9718C⬎T c.10174C⬎T c.11612G⬎A c.126delT c.498delT c.711_714delAATG c.1068_1069insT c.1106_1113delGACAACCTinsTGTC c.1409_1410delGT c.1620_1621insAGTT c.1624delTTAC c.3306delT c.3761_3762delCCinsG c.3958_3959delGG c.5378_IVS⫹1delATGG c.5895_5896insA

R328X R496X R2671X R3107X R3240X Q3392X W3871X F42fsX63 T166fsX178 S237fsX244 P356fsX357 G369fsX412 G470fsX480 E541fsX556 L542fsX545 Y1102X A1254fsX1302 G1320fsX1322 D1793fsX1802 L1966fsX1969

Ex57 Ex58 Ex60 Ex61 Ex61

c.8829_8830insC c.9347delT c.10075delG c.10239delA c.10481_10503delTTCTC TTGGCTGTATTCTACCAT c.10637delT c.10972_10973delAT IVS13-1G⬎A IVS16⫹1G⬎A

I2944fsX2949 L3116fsX3161 G3359fsX3399 L3413fsX3432 L3494fsX3528

Ex61 Ex61 IVS13 IVS16

V3546fsX3567 I3658fsX3664

frameshift mutation near the C-terminus (I3658fsX3664), supposedly removing the unique transmembrane domain and the cytoplasmic tail while leaving most of the extracellular portion alone. It is thus suggested that a critical amount of full-length polyductin is required for a proper protein function. Given the multitude of missense alleles and the variety in phenotypic presentation, patients with PKHD1 mutations will be an interesting population to study genotype-phenotype correlations. This article provides the first clues that some missense mutations are associated with a uniform phenotype (see Results section). However, more sustainable genotype-phenotype correlations will require identification of further patients harboring a peculiar genotype. Interfamilial phenotypic variability is common in ARPKD and presumably based on multiple allelism. In a given family, the clinical course among affected siblings is usually comparable (3,30); however, a small proportion of sibships included in the present study exhibited marked intrafamilial clinical variability (Table 2). Obviously, phenotypes caused by PKHD1 mutations cannot be explained on the basis of the genotype alone, but supposedly also

Mouse

Reference Present study Present study (18x) Ward et al. Present study (2x) Present study Ward et al. Ward et al. Present study Present study Onuchic et al. Present study (2x) Present study Present study Onuchic et al. Ward et al. Onuchic et al. Onuchic et al. Present study Present study Ward et al. Onuchic et al. (3x) Present study (2x) Onuchic et al. Present study Onuchic et al. Present study Present study Ward et al. Present study Present study Present study

Origin Turkey Finland Turkey/ Syria Italy

Germany Denmark UK Turkey Portugal US Germany Turkey US Inuit (Canada) Turkey US (2x)/UK France Afrikaner Germany Turkey Germany UK

Turkey Finland Israel Arab

depend on the background of other genes, epigenetic factors, and environmental influences. It might be interesting to determine whether modifier genes that influence disease severity in mouse models for polycystic kidney disease modulate the phenotype in human ARPKD (31,32). Moreover, among epigenetic factors, the process of alternative splicing might be relevant as a modifying mechanism peculiarly in the setting of multiple putative polyductin splice variants (33,34).

Advantages and Limitations of Direct Mutation Analysis Due to the poor prognosis of early manifestations of ARPKD, many parents seek prenatal diagnosis. Thus far, without knowing the PKHD1 gene, prenatal diagnosis was only feasible by indirect genotyping. However, interpretation of haplotype-based analysis might prove difficult in cases without an unambiguous clinicopathologic diagnosis. Notably, in three families from the present study with identified mutations (families 264, 385, and 427), early manifesting autosomal dominant polycystic kidney disease had to



87

Table 4. Continued Type of Mutation

Location

Missense

Ex3

c.107C⬎T

aa Change T36M

Mouse T37

c.667G⬎A c.757T⬎C c.1418T⬎G c.1487G⬎C c.1968G⬎T c.2107G⬎A c.2279G⬎A c.2414C⬎T c.2990T⬎A c.3089C⬎A c.3364G⬎A c.3367G⬎A c.3747T⬎G c.4220T⬎G c.4457C⬎T c.4870C⬎T c.4991C⬎T c.5221G⬎A c.5750A⬎G c.5984A⬎G c.5993T⬎C c.6401T⬎C c.6992T⬎A

G223S F253L I473S R496P W656C D703N R760H P805L M997K A1030E G1122S G1123S C1249W L1407R P1486L R1624W S1664F V1741M Q1917R E1995G I1998T L2134P I2331K

G221 F251 I471 R494 W654 D701 C758 P803 M995 A1028 G1120 G1121 C1247 L1403 P1482 Q1620 S1660 V1737 Q1913 E1991 I1994 L2129 M2326

Ex52 Ex57

c.8281G⬎T c.8870T⬎C

D2761Y I2957T

D2755 I2951

c.9053C⬎T c.9241A⬎G c.9415G⬎T c.9878A⬎T c.10412T⬎G c.10444C⬎T c.10658T⬎C

S3018F I3081V D3139Y D3293V V3471G R3482C I3553T

Reference Ward et al. (2x) Onuchic et al. Present study (9x)

Ex9 Ex11 Ex16 Ex16 Ex21 Ex21 Ex22 Ex24 Ex27 Ex27 Ex29 Ex30 Ex32 Ex32 Ex32 Ex32 Ex32 Ex32 Ex35 Ex37 Ex37 Ex39 Ex43

Ex58 Ex58 Ex58 Ex59 Ex61 Ex61 Ex61 a

cDNA Change

S3012 I3075 N3132 D3286 V3464 R3475 V3546

Onuchic et al. (2x) Present study Present study Onuchic et al. Present study Present study Present study Present study Onuchic et al. Present study Present study Present study Onuchic et al. Present study Ward et al. Ward et al. Present study Onuchic et al. (3x) Ward et al. Ward et al. Ward et al. Ward et al. Present study Present study Ward et al. Present study (2x) Present study Ward et al. Onuchic et al. Present study Ward et al. Present study Onuchic et al. Present study Present study (5x) Present study (4x) Ward et al.

Origin

US UK / Finland (3x) / Germany (4x) US / Afrikaner Italy Pakistan US Turkey Turkey Germany Netherlands Saudi-Arabia UK Germany Germany US Germany

Germany Saudi-Arabia

Turkey France Finland Finland US Finland France Germany Turkey Finland Germany (2x) / Israel Arab (2x)

In the setting of parental consanguinity, mutations were counted only once per family.

be considered because parental renal or hepatic ultrasound revealed areas suspicious of cyst formations (35). Thus, in families with diagnostic uncertainties, characterization of PKHD1 mutations allows to offer accurate genetic counseling and prenatal diagnosis. However, mutation analysis in ARPKD poses special difficulties due to the huge size of the gene, multiple predicted splice variants, and marked allelic heterogeneity. Special note is warranted regarding difficul-

ties in differentiating pathogenic nucleotide substitutions from harmless sequence variants. Due to the aforementioned challenges and limitations of PKHD1 mutation screening, haplotype determination of closely flanking and intragenic microsatellites will be further used for efficient genotyping in families without diagnostic doubts. However, in families with diagnostic challenges or families in whom no DNA of the oftentimes deceased index patient is available, a direct

88


Figure 3. Haplotype analysis and the identified PKHD1 mutation in family 2. The affected child 2.1 was found to be heterozygous for microsatellite markers from the ARPKD region while homozygously carrying the T36M mutation in the PKHD1 gene.

approach will be the only option to establish a molecular genetic diagnosis.

Acknowledgments The authors would like to thank the patients and families as well as their physicians who were involved in these studies for their cooperation. The technical assistance of Christiane Lupczyk, Edith von Heel, and Edith Bu¨ nger is gratefully acknowledged. This work was supported by the Deutsche Forschungsgemeinschaft and by National Institutes of Health grant R01 DK51259.

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4. Zerres K, Hansmann M, Mallmann R, Gembruch U: Autosomal recessive polycystic kidney disease. Problems of prenatal diagnosis. Prenat Diagn Mar 8: 215–229, 1988 5. Reuss A, Wladimiroff JW, Stewart PA, Niermeijer MF: Prenatal diagnosis by ultrasound in pregnancies at risk for autosomal recessive polycystic kidney disease. Ultrasound Med Biol 16: 355–359, 1990 6. Zerres K, Mu¨ cher G, Becker J, Steinkamm C, Rudnik-Schoneborn S, Heikkila P, Rapola J, Salonen R, Germino GG, Onuchic L, Somlo S, Avner ED, Harman LA, Stockwin JM, Guay-Woodford LM: Prenatal diagnosis of autosomal recessive polycystic kidney disease (ARPKD): Molecular genetics, clinical experience, and fetal morphology. Am J Med Genet 76: 137–144, 1998 7. Roy S, Dillon MJ, Trompeter RS, Barratt TM: Autosomal recessive polycystic kidney disease: long-term outcome of neonatal survivors. Pediatr Nephrol 11: 302–306, 1997 8. Zerres K, Mu¨ cher G, Bachner L, Deschennes G, Eggermann T, Kaariainen H, Knapp M, Lennert T, Misselwitz J, von Muhlendahl KE: Mapping of the gene for autosomal recessive polycystic kidney disease (ARPKD) to chromosome 6p21-cen. Nat Genet 7: 429 – 432, 1994 9. Ward CJ, Hogan MC, Rossetti S, Walker D, Sneddon T, Wang X, Kubly V, Cunningham JM, Bacallao R, Ishibashi M, Milliner DS, Torres VE, Harris PC: The gene mutated in autosomal recessive polycystic kidney disease encodes a large, receptor-like protein. Nat Genet 30: 259 –269, 2002 10. Onuchic LF, Furu L, Nagasawa Y, Hou X, Eggermann T, Ren Z, Bergmann C, Senderek J, Esquivel E, Zeltner R, RudnikSchoneborn S, Mrug M, Sweeney W, Avner ED, Zerres K, Guay-Woodford LM, Somlo S, Germino GG: PKHD1, the polycystic kidney and hepatic disease 1 gene, encodes a novel large protein containing multiple immunoglobulin-like plexin-transcription-factor domains and parallel beta-helix 1 repeats. Am J Hum Genet 70: 1305–1317, 2002 11. Lager DJ, Qian Q, Bengal RJ, Ishibashi M, Torres VE: The pck rat: A new model that resembles human autosomal dominant polycystic kidney and liver disease. Kidney Int 59: 126 –136, 2001 12. Sanzen T, Harada K, Yasoshima M, Kawamura Y, Ishibashi M, Nakanuma Y: Polycystic kidney rat is a novel animal model of Caroli’s disease associated with congenital hepatic fibrosis. Am J Pathol 158: 1605–1612, 2001 13. Moser M, Pscherer A, Roth C, Becker J, Mu¨ cher G, Zerres K, Dixkens C, Weis J, Guay-Woodford L, Buettner R, Fassler R: Enhanced apoptotic cell death of renal epithelial cells in mice lacking transcription factor AP-2beta. Genes Dev 11: 1938 – 1948, 1997 14. Onuchic LF, Mrug M, Lakings AL, Mu¨ cher G, Becker J, Zerres K, Avner ED, Dixit M, Somlo S, Germino GG, Guay-Woodford LM: Genomic organization of the KIAA0057 gene that encodes a TRAM-like protein and its exclusion as a polycystic kidney and hepatic disease 1 (PKHD1) candidate gene. Mamm Genome 10: 1175–1178, 1999 15. Hofmann Y, Becker J, Wright F, Avner ED, Mrug M, GuayWoodford LM, Somlo S, Zerres K, Germino GG, Onuchic LF: Genomic structure of the gene for the human P1 protein (MCM3) and its exclusion as a candidate for autosomal recessive polycystic kidney disease. Eur J Hum Genet 8: 163–166, 2000 16. Onuchic LF, Mrug M, Hou X, Nagasawa Y, Furu L, Eggermann T, Bergmann C, Mu¨ cher G, Avner ED, Zerres K, Somlo S, Germino GG, Guay-Woodford LM: Refinement of the autosomal recessive polycystic kidney disease (PKHD1) interval and exclu-


17.

18.

19.

20.

21.

22.

23.

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