Am. J. Hum. Genet. 54:757-764, 1994

Congenital Nephrotic Syndrome of the Finnish Type Maps tothe Long Arm of Chromosome 19Marjo Kestila,*,* Minna Mannikko,* Christer Holmberg,s Gabor Gyapay,11 Jean Weissenbach,11Eeva-Riitta Savolainen,t Leena Peltonen,1 and Karl Tryggvason*

*Biocenter and Department of Biochemistry, and tDepartment of Clinical Chemistry, University of Oulu, Oulu; *Department of HumanMolecular Genetics, National Public Health Institute and lChildren's Hospital, University of Helsinki, Helsinki; lGenethon, Evry

Summary

Congenital nephrotic syndrome of the Finnish type (CNF) is an autosomal recessive disease that is characterizedby massive proteinuria and nephrotic syndrome at birth. CNF represents a unique, apparently specificdysfunction of the renal basement membranes, and the estimated incidence of CNF in the isolated populationof Finland is 1 in 8,000 newborns. The basic defect is unknown, and no specific biochemical defect or

chromosomal aberrations have been described. Here we report the assignment of the CNF locus to 19ql2-ql3.1on the basis of linkage analyses in 17 Finnish families. Multipoint analyses and observed recombination eventsplace the CNF locus between multiallelic markers D19S416 and D19S224, and the significant linkagedisequilibrium observed suggests that the CNF gene lies in the immediate vicinity of the markers D19S224 andD19S220.

Introduction

Congenital nephrotic syndrome of the Finnish type(CNF; MIM 256300) is inherited as an autosomal re-cessive trait displaying an incidence of 12.2 in 100,000newborns in Finland (Huttunen 1976). The disease isrepresentative of about 30 diseases found enriched inthis genetically isolated population. CNF represents aunique biological model for kidney dysfunction, thedisease being characterized by massive proteinuriastarting in utero, an enlarged placenta weighing over25% of the child's birth weight, and early manifestationof signs of nephrosis, usually at birth (Hallman et al.1967). CNF can be diagnosed prenatally by analyses ofthe a-fetoprotein levels in the amniotic fluid and ma-ternal serum (SeppilA et al. 1976), and this method hasbeen used especially in high-risk families (Aula et al.1978). The disease is progressive during the first 1-2

Received October 19, 1993; accepted for publication December23, 1993.

Address for correspondence and reprints: Leena Peltonen, Depart-ment of Human Molecular Genetics, National Public Health Insti-tute, Mannerheimintie 166, FIN-00300 Helsinki, Finland.© 1994 by The American Society of Human Genetics. All rights reserved.0002-9297/94/5405-0004$02.00

years of life, and kidney transplantation is the only suc-cessful and life-saving treatment for CNF patients(Holmberg et al. 1991). Histopathological findingsfrom CNF kidneys typically include dilated proximaltubules, areas of tubular atrophy, interstitial fibrosis,mesangial cell proliferation, and sclerosis and fibrosisof the glomeruli. Loss of the foot processes of the glo-merular epithelial cells are the usual findings in electronmicroscopy, but no abnormalities are apparent in theglomerular basement membrane (GBM), which func-tions as the filtration barrier for macromolecules (Hall-man et al. 1973; Aula et al. 1978).

In spite of intensive research efforts, the biochemicaldefect leading to CNF is unknown. Because of the pres-ence of massive proteinuria, it has been hypothesizedthat the disease is caused by a mutation in a gene codingfor either a structural basement membrane componentor a component crucial for the filtration process. How-ever, using linkage analyses, we have excluded sevenautosomal genes encoding the structural proteins typeIV collagen and laminin as CNF loci. Furthermore, thegene encoding the core protein of perlecan, a heparansulfate proteoglycan thought to play a role in the filtra-tion process (Caulfield and Farquhar 1978), and thePax-2 gene, which encodes a transcription factor im-

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portant for kidney development (Dressler et al. 1993),are not linked to CNF (KestilA et al., in press-a, inpress-b).

Since CNF has not been linked to any potential can-didate gene, we have applied the random mapping ap-proach, which utilizes highly informative markers as-signed to individual chromosomes to map the CNFgene. Here we report the assignment of the CNF locusto a defined chromosomal region on 19ql2-ql3.1

Material and Methods

Family MaterialThe family material consisted of 17 Finnish CNF fam-

ilies, each with one or two affected individuals and be-tween one and nine healthy siblings, for a total of 86individuals. The diagnosis of CNF was based on severeproteinuria, a large placenta (>25% of birth weight),nephrotic syndrome during the first weeks of life, andexclusion of other types of congenital nephrotic syn-drome (Koskimies 1990). Renal histology typical ofCNF was subsequently documented at nephrectomyprior to renal transplantation.

DNA SamplesCell lines of skin fibroblasts or lymphocytes immor-

talized by Epstein-Barr virus were established, and geno-mic DNA was extracted from cultured cells by usinganautomated Applied Biosystems 340A extractor.

Detection of PolymorphismsPolymorphic microsatellite markers were analyzed

by using PCR and polyacrylamide gel electrophoresis.PCR was performed in 15 Rl containing 10 ng of tem-plate DNA, 3 pmol of primers, 0.2 gM of each nucleo-tide, 20 mM Tris-HC1 (pH 8.8), 15 mM (NH2)SO4, 1.5mM MgCl2, 0.1% Tween 20, 0.01% gelatin, and 0.25 UThermus aquaticus DNA polymerase (Promega). Oneof the primers was labeled at the 5' end using 32P-yATP.The PCR reactions were performed in multiwell micro-titration plates for 26 cycles with denaturation at 940Cfor 30 s, annealing at different temperatures, dependingon the primers, for 30 s, and extension at 72°C for 30 s.In the first cycle, denaturation was carried out for 4min, and extension in the last cycle was for 5 min. Theamplified fragments were separated using 5% PAGE.Most of the markers originated from the Genethon am-plifiable marker collection (The Genethon Microsatel-lite Map Catalogue 1992) and the Nordic HUGOmarker collection.

Linkage AnalysisComplete penetrance of the disease was assumed for

the analyses, because the age at onset of the disease isduring the first months of life. On the basis of the factthat CNF is a typical representative of the Finnish dis-ease heritage, we assumed that the possibility of locusheterogeneity could be ignored. Linkage analyses wereperformed with the MLINK and LINKMAP options ofthe Linkage package (version 5.10) (Lathrop et al.1984). X2 tests were performed with the CONTINGoption of LINKUTIL, and the EH option was used toestimate haplotype frequencies of markers with linkagedisequilibrium. We performed multipoint LINKMAPanalyses, which adapt linkage disequilibrium and com-bined information of markers D19S224 and D19S220,and used that combination as one marker demonstrat-ing a deviation in allelic frequencies in the CNF chro-mosomes. The D19S416 marker was incorporated intothe haplotype frequencies, assuming equilibrium, be-cause it did not demonstrate linkage disequilibrium. Adisease gene frequency of .01 was used for the haplo-type frequencies, which were determined for each mapinterval, depending on the location of the CNF locus.

Results

Linkage AnalysisA total of 90 highly informative markers randomly

spread in all autosomes were analyzed in our familymaterial until highly promising lod scores were ob-served with markers localized to the long arm of chro-mosome 19. Consequently, more markers were ana-lyzed in this region, and the highest maximal lod scoreswere observed with markers D19S49 (Mfdll), D19S75(Mfdl3), D19S225 (AFM248zc1), D19S416 (AFM304xf5),and D19S224 (AFM240vc1). The lod scores obtainedin pairwise linkage analyses between CNF and chromo-some 19 markers are shown in table 1, and the orderand the estimated distances (0) between the analyzedmarkers are shown in figure 1. The order and the dis-tances of the markers (sex-average) are based on recom-binations observed in our material and on unpublishedinformation provided by one of the authors (J. Weis-senbach). Since no recombinations have been observedbetween numerous markers, the order of these cannotbe unequivocally determined, but this has no signifi-cance on the data obtained in the linkage analyses.The observed obligatory recombination events with

13 chromosome 19 markers in the three most informa-tive CNF families are presented in figure 2. On the basisof the recombinations observed in families 9 and 11,

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CNF Maps to the Long Arm of Chromosome 19

Table I

Results of Two-Point Linkage Analyses between CNF and Chromosome 19 Markers

LOD SCORE (Z) AT 0 =90%

.00 .01 .05 .10 .20 .30 .40 Zmax 0max CONFIDENCE LIMITS

D19S409 ...... c -.51 .09 .22 .19 .09 .02 .23 .11D19S49 ....... 2.97 2.84 2.35 1.79 .92 .38 .09 2.97 .00 0;.08D19S75 ....... 3.45 3.33 2.88 2.32 1.29 .54 .13 3.45 .00 0;.08D19S225 ...... -00 3.19 3.16 2.60 1.44 .60 .15 3.31 .02 0;.12D19S208 ...... 2.24 2.17 1.88 1.52 .86 .38 .10 2.24 .00 0;.14D19S191 ...... -cc 2.08 2.22 1.85 1.02 .42 .10 2.28 .03 0;.16D19S213 ...... -00 1.34 1.60 1.39 .80 .33 .08 1.61 .04 0;.23D19S425 ...... 2.45 2.37 2.08 1.71 1.04 .50 .13 2.45 .00 0;.13D19S416 ...... -cc 2.91 2.97 2.50 1.44 .63 .16 3.07 .03 0;.13D19S224 ...... -cc 2.61 2.67 2.22 1.22 .48 .11 2.77 .02 0;.14D19S220 ...... -cc 1.57 1.78 1.51 .86 .38 .09 1.80 .02 0;.20D19S422 ...... -0C -.51 .57 .76 .60 .32 .09 .76 .10D19S223 ...... -cc -.30 1.19 1.35 .93 .43 .11 1.36 .09

the CNF locus could be narrowed down to a regioneither between markers D19S409 and D19S225 or be-tween markers D19S416 and D19S224. When informa-tion from these markers was combined in a multipoint

D198409

p13. 1

q13.1I

D19S75

D19S225

D19S208

D19S191

D19S213

D198425

D19S416

D19S224

D19S220

D19S422

D198223

0.00

0.03

0.01

0.01

0.00

0.00

0.00

0.00

0.03

0.00

:1 0.01

-1 0.03

19

Figure I The utilized markers and their estimated order anddistances (i.e., 0) on chromosome 19q12-q13.1.

LINKMAP analysis (fig. 3), the highest lod score, 4.58,was observed in the position between markersD19S416 and D19S224, suggesting that the CNF geneis located in the 3-cM chromosomal region flanked bythese two markers. The relative likelihoods of the locusposition have been presented for each interval. In theLINKMAP analysis we adapted the marker distancespresented in figure 1. The alleles observed in CNF fami-lies with markers D19S416 and D19S224 are shown infigure 4. No evidence for locus heterogeneity was ob-served in the Finnish CNF families, neither in two-point nor in multipoint linkage analyses.

Linkage DisequilibriumSince linkage disequilibrium is an exceptionally pow-

erful tool for determining the position of disease loci inisolated populations with a high probability of one an-cient mutation, we tested for possible linkage disequi-librium with linked markers in CNF chromosomes byusing the X2 test. Markers D19S224 and D19S220 werefound to show significant linkage disequilibrium, witha value of P < .001. The observed alleles with thesemarkers are shown in figure 4, and the distribution ofthe alleles in CNF and non-CNF chromosomes, as wellas the results of the X2 test are shown in table 2. Withmarker D19S224, allele 7 showed a 66% allelic associa-tion to CNF chromosomes, and with marker D19S220,allele 8 showed a 69% allelic association. Analysis ofhaplotypes generated with markers D19S224 andD19S220 revealed that eight different haplotypes canbe observed in CNF chromosomes and 25 haplotypes

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S409 S75 S208 S213 8416 8220 S223

9 E)TO 849 8225 S191 S425 S224 8422

6--56 dd *6bibbb lAPh 0 *00 0 0

11~I

1i

*eoooeee0000e

.o00o.000o00o

0 no recombination * recombination * uninformative melosim

Figure 2 Observed recombination events in the three most informative families. On the basis of these recombinations, the CNF locuscould lie between markers D19S409 and D19S225 or between D19S416 and D19S224.

in non-CNF chromosomes. Sixty-three percent of CNFchromosomes carried haplotype 7-8, which was notseen in any non-CNF chromosomes (table 3).When the observed linkage disequilibrium was

adapted for linkage analyses, the power of the linkage

Lod score 4(Z) +12-.

-2'

-3-

analyses and consequently the significance of the ob-tained lod scores increased substantially. Results oftwo-point linkage analyses adapting the linkage disequi-librium observed with markers D19S224 and D19S220are displayed in table 4. We also allowed for linkage

IIII

N

IIIIIIIIII**\I

Figure 3 Multipoint linkage analysis in 17 Finnish CNF families, on chromosome 19. D19S224 was arbitrarily placed at 0. The relativelikelihoods of the locus position have been presented for each interval. Recombination events were not observed between markers D19S208,D19S191, D19S213, D19S425, and D19S416, but in LINKMAP analysis the recombination fractions were set to .0001, for practical reasons.The situation is analogous with D19S224 and D19S220 and with D19S409 and D19S49. The solid line indicates multipoint analysis assumingequilibrium; the dashed line indicates linkage disequilibrium included.

760

I

CNF Maps to the Long Arm of Chromosome 19

D)196416D19S224 6-7 5-67-5 ~~7-3D19S220 8-3 3 8-8

7-5 6-5 7-5 7-5 6-6 6-6 6-5 7-5 6-6 6-65-7 7-3 5-7 5-7 7-3 7-3 7-7 5-7 7-3 7-33-8 8-8 3-8 3-8 8-8 8-8 8-8 3-8 8-8 8-8

8-6 19 4-67-4 4-5

8-5 6-6

n.d. 6-6 8-4 8-4n.d. 4-5 7-4 7-4n.d. 5-6 8-6 8-6

6-6 11 8-3

7-7 7-10

8-11 8-3

6-8 6-87-7 7-108-8 8-3

7-6 4-7 4-6 7-7 4-66-7 7-7 7-7 6-7 7-75-8 8-5 8-8 5-5 8-8

5-7 8 5-77-9 7-28-5 | -6

7-5 5-7 5-5 7-59-7 7-2 7-7 9-75-8 8-6 8-8 5-8

9-6 7 2-1 6-6 1 2-6 5-6 1 6-7 8-10 6-79-5 7-9 4-3 73 4-547 79 -

9-2) 84 25 (8-42725-5 43

9-2 6-1 6-1 6-6 6-2 6-6 6-6 5-6 5-7 8-6 10-7 10-79-7 5-7 5-7 4-3 4-7 4-3 5-4 4-4 4-7 7-5 9-1 9-19-8 2-8 2-8 2-4 2-8 2-4 7-2 2-2 2-5 8-4 5-3 5-3

6-617-98-7

8-9 7-76-7 8-8

3-97-78-6

7-7 7-76-6 6-8

6-3 G 5-8 6-7 17 n d.7-9 7-9 79648-5 l_ B-3 8-4 I[t 8-8

3-8 6-5 7-7 6-69-9 7-7 9-4 7-65-3 8-8 4-8 8-8

1-2 1 6-6 6-7 2 6-2 6-6 1 1-7 7-7 2-7

4-3 4-9 7-4 .....I7-4 7- [3l 9 7-4 4-8

2-6 2-6 8-4 [I 8-6 8-6 8-6 8-3 2-4

2-6 1-6 6-6 7-6 6-1 6-7 7-2 7-73-4 4-4 7-7 4-7 7-3 7-9 7-4 4-86-2 2-2 8-8 4-8 8-8 8-6 8-2 3-4

Figure 4 The observed alleles with markers D19S416, D19S224, and D19S220 in 17 Finnish CNF families. An asterisk (*) indicates thatno sample was available; a double asterisk (**) indicates unknown affection status.

disequilibrium in multipoint LINKMAP analysis, inwhich we used combined information from markersD19S224 and D19S220. The results of this LINKMAPanalysis, which incorporates the linkage disequilibrium,are represented by a dashed line in figure 3.To further limit the critical chromosomal region for

CNF we adapted the Luria-DelbrUck formula that wasoriginally developed for bacterial populations (Luriaand DelbrUck 1943) and later applied to disease genesenriched in isolated populations (Histbacka et al.1992). This formula hypothesizes one ancient mutationand utilizes the linkage disequilibrium to estimate theprobable distance between the disease gene and themarker demonstrating enrichment in disease alleles.When this formula was adapted to our CNF materialwe could approximate that the CNF gene is located

about 350-400 kb from markers D19S224 andD19S220, which could not be separated on the ge-netic map.

DiscussionIn the present study, the gene locus defective in CNF

was assigned to the chromosomal region 19q12-q13.1.The chromosomal localization is reported only for oneof the markers linked to CNF, D19S49, which is as-signed to 19q12 (NIH/CEPH Collaborative MappingGroup 1992). Two markers in this region revealed sta-tistically significant linkage disequilibrium in FinnishCNF families, and when the linkage disequilibrium wasadapted for linkage analyses, the lod scores increasedsignificantly. Combined information from the multi-point linkage and linkage disequilibrium analyses

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Table 2

Distribution of Alleles In D19S224 and Dl 9S220 in CNFChromosomes and Non-CNF Chromosomes

CNF Non-CNF

D19S224 alleles:'1 ........... 0 12 ............. 0 13 ............. 1 44 ............. 7 55 ............. 1 46 ............. 1 17 ............. 21 48 ............. 0 29 .......... 1 10job .................... 0 0

Total ....... 32 32D19S220 alleles:c

lb ..................... 0 02 ............. 6 13 ............. 0 44 ............. 1 55 ............. 0 96 ............. 2 97 ............. 0 28 ............. 22 29 .......... 1 0

Total ....... 32 32

a x2 = 26.86; df = 8; P = .000748.b Alleles are observed only in the recombination family, and they

are not included in the test for linkage disequilibrium.cX2 = 43.36; df = 7; P = .000000.

placed the CNF gene between markers D19S416 andD19S224/D19S220, closer to D19S224/D19S220,with the critical chromosomal region thus being 2-3 cM.

Since CNF represents a disease enriched in the geneti-cally isolated population of Finland, one can hypothe-size that one ancient mutation has spread throughoutthe population in subsequent generations. This allowsthe use of the Luria-Delbriick formula, originally devel-oped for the estimation of genetic distances in bacterialpopulations, to approximate the most probable dis-tance between the disease locus and markers demon-strating the linkage disequilibrium. This approximationwould place the CNF locus within 400 kb of the closestmarkers, a distance that could be covered by using thetechniques of molecular cloning. The goal for the imme-diate future is to identify more polymorphic markers inthe D19S224/D19S220 region in order to reveal a moresignificant allelic association with the disease and toprovide the possibility to define the position of theCNF locus with greater accuracy.

The present results are of significance for several rea-sons. First, they are of clinical importance because theywill immediately facilitate the development of genetictests for CNF families, and, second, the isolation of thegene may provide new insight into the exploration ofkidney physiology and development. A DNA-baseddiagnostic test will have significant advantages over thea-fetoprotein assay that is currently used for prenataldiagnosis of CNF. The latter can only be carried outafter the 15th gestation week, and increased levels ofa-fetoprotein are not specific for CNF but can also beobserved in other fetal abnormalities, such as spina bi-fida and other types of proteinuria that start in utero.Furthermore, this test cannot be used for carrierscreening. In contrast, a DNA test is specific and can beused both for prenatal diagnosis of chorionic villus sam-ples as early as during the first trimester and for carrierscreening from blood samples. When an additional in-formative marker(s) is found in this region, it will bepossible to implement a DNA method for prenataldiagnosis and even carrier screening of high-risk fami-lies, until the actual gene defects have been identified.More important tasks, however, are to identify thegene defective in CNF and to determine the nature ofthe mutations in CNF kindreds. This will be requiredfor precise DNA diagnosis and will have consequencesfor the development of gene therapy. At present, thereare no known candidate genes on 19q12-q13.1. Noneof the known basement membrane genes has been local-ized to this chromosome, and this region does not con-tain any other potential candidate genes coding for mo-lecular components of the kidney.

Table 3

Distribution of DI9S224-DI 9S220 Haplotypes in CNFChromosomes and Non-CNF Chromosomes

No. (%) OF HAPLOTYPES IN

CNF Chromosomes Non-CNF Chromosomes

3-8 .............. 1 (3) 1 (3)4-2 .............. 6 (19) 0 (0)4-6 .............. 1 (3) 1 (3)5-4 .............. 1 (3) 0 (0)6-8 .............. 1 (3) 0 (0)7-6 .............. 1 (3) 1 (3)7-8 .............. 20 (63) 0 (0)9-9 .............. 1 (3) 0(0)22 Haplotypes 0 (0) 29 (91)60 Haplotypes 0 (0) 0 (0)

Total 32 32

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CNF Maps to the Long Arm of Chromosome 19 763

Table 4

The Results of Two-Point Linkage Analyses with Adapted Linkage Disequilibrium

LOD SCORE (Z) AT 0 =

.00 .01 .05 .10 .20 .30 .40 Z.,mx Omax 90% CONFIDENCE LIMrIS

D19S224 .... -00 8.47 8.29 7.51 5.68 3.81 1.94 8.55 .02 0;.09D19S220 ...... -OO 10.05 9.96 9.23 7.34 5.17 2.74 10.16 .02 0;.10

As a consequence, the CNF gene will most likelyhave to be isolated by positional cloning. This task mayactually be quite feasible at this stage, as work on thephysical map of chromosome 19 is advancing rapidly.For example, a cytogenetic band location of >500 cos-mids and 70 genes or DNA markers was recently re-ported for this chromosome (Trask et al. 1993). Itseems likely that YAC or cosmid contigs will soon beavailable for the region to which the CNF gene wasassigned in this study.The cloning of the CNF gene is intriguing because it

can provide new information about the still poorly un-derstood mechanisms of glomerular filtration. The mo-lecular basis for this process has been one of the centralquestions of kidney research. The lack of extrarenalsymptoms in CNF and the massive proteinuria ob-served even during embryonic development impliesthat the gene defect may involve a GBM componentthat is crucial for kidney filtration. Another possibilityis that the gene is important for glomerular and tubulardevelopment. In support of the latter possibility is arecent study (Dressler et al. 1993) showing that trans-genic mice with a deregulated Pax-2 gene, which en-codes a transcription factor important for renal devel-opment, exhibit a congenital nephrotic syndrome withfeatures similar to those found in CNF patients. Theisolation of the human CNF gene will enable interestingstudies on animal models, in order to study this aspect.

AcknowledgmentsWe would like to thank the CNF families who participated

in this study. We also thank L. Ollitervo, T. Vakiparta, and S.Johansson for their expert technical assistance; P. Tienari andJ. Terwilliger for their help with the linkage analyses; N. P.Huttunen, P. Lautala, and M. Poyhonen for providing patientsamples; and The Nordic HUGO Resource for the amplifi-able markers. The efficient help of Sophie Parc in providingthe information on Genethon markers is appreciated. Thiswork was supported in part by grants from The Sigrid Juselius

Foundation, The Academy of Finland, and The FinnishCancer Institute.

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