Location of the first genetic locus, PKDr1, controlling autosomal dominant polycystic kidney disease in Han:SPRD cy/+ rat
Location of the first genetic locus, PKDr1, controlling autosomal dominant polycystic kidney disease in Han:SPRD cy/+ ratMarie-ThérèseBihoreau*, IsabellaCeccherini1, JulieBrowne, BetinaKränzlin2, GiovanniRomeo1,3, G. MarkLathrop, Michael R.James and NorbertGretz2,4
The Wellcome Trust Centre for Human Genetics, University of Oxford,Oxford OX3 7BN,UK,1Laboratory of Molecular Genetics, Istituto Gaslini, 16148Genoa,Italy,2Medical Research Center, University of Heidelberg, Klinikum Mannheim, D-68167Mannheim,Germany,3Genetic Cancer Susceptibility Unit, International Agency for Research on Cancer, 69372Lyon,France and4V. Medical Department, University of Heidelberg, Klinikum Mannheim, D-68167Mannheim,Germany
Recieved December 17, 1996;Revised and Accepted January 23, 1997
The Han:SPRD cy/+ strain develops a form of slowly progressive disease that appears similar in many respects to that seen in the autosomal dominant polycystic kidney disease (ADPKD) in humans. We have performed a total genome scan in an experimental backcross population derived from affected Han:SPRD cy/+ rat (PKD) and non-affected Wistar Ottawa Karlsburg rat (WOK) using 117 microsatellite markers. The genetic dissection of PKD allowed us to map on rat chromosome 5, a quantitative trait locus (QTL) controlling PKD, kidney mass and plasma urea concentration. The homology region is likely to reside on human chromosome 8. The gene responsible for PKD in Han:SPRD cy/+ rat is neither PKD1, localised on human chromosome 16, nor PKD2, localised on human chromosome 4. Therefore, we propose that this new locus be denoted PKDr1. The detection of the PKDr1 locus and associated QTL should accelerate research into the genetic causes of ADPKD.
Autosomal dominant polycystic kidney disease (ADPKD) is one of the most common monogenic diseases in humans, affecting 1 in 1000 individuals. This disorder typically shows a slow progression in which renal cysts increase in number and size, gradually leading to renal insufficiency which may require dialysis and/or renal transplantation. Other organs, such as the liver, can be affected by the invasion of cysts, and hypertension may also be a symptom of the disease (1 ). Two genes implicated in the disease, PKD1 and PKD2, localised on human chromosome 16 and chromosome 4 respectively, have been identified through positional cloning (2 ,3 ). However, in some families, ADPKD segregates independently of either PKD1 or PKD2, demonstrating the involvement of other loci (4 ,5 ).
Due to the genetic complexity of ADPKD in humans, numerous animal models of polycystic kidney disease have been described, most of them being mouse models with only one well-documented rat model available (6 ). The available mouse models suffer several drawbacks for the study of polycystic kidney disease. Firstly, the mice generally model the recessive form of polycystic kidney disease. In addition, affected mice have a short survival period (a few weeks) and thus rarely live long enough for suitable treatment to be undertaken. The small size of the mice adds to the difficulties in describing functional parameters of the progression of the disease, i.e. blood pressure and blood borne parameters of renal function such as urea concentration. In contrast to mouse models, a well-documented rat model of autosomal dominant polycystic kidney disease is available: the Han:SPRD cy/+ rat model (7 -11 ). The different features of the Han:SPRD cy/+ strain (PKD) and the similarities between rat and human PKD are listed in Table1 . Although inherited as a simple Mendelian disorder, an effect of the genetic background on the disease expression in the Han:SPRD cy/+ rat is postulated analogous to that already demonstrated in the mouse, where several modifier genes have been mapped to different locations in the mouse genome (12 -14 ). The action of different genes has also been proposed to explain some still undefined aspects of human PKD, such as early onset (15 ). On the basis of these observations it is reasonable to consider rat PKD as a complex disease.
Recent progress to increase the density of the rat genetic linkage map (16 ,17 ) allows chromosome mapping of gene(s) responsible for disease in rat models for human genopathy. The current study has been designed to perform a total genome scan for factors controlling the disease in an experimental backcross population derived from affected Han:SPRD cy/+ rat (PKD) and non-affected Wistar Ottawa Karlsburg rat (WOK). Consequently, we detected a locus responsible for PKD on rat chromosome 5, and we were able to exclude the candidate regions of rat chromosome 10 and 14, homologous to the human PKD1 and PKD2 regions respectively. Thus, PKD in the Han:SPRD cy/+ rat appears to be due to a mutation in a novel gene, the identification of which may give new insights into the disease.
A cohort of 91 male (PKD*WOK) backcross 1 hybrid rats (BC1) was studied; 59 originated from affected PKD males and non-affected females and 32 from PKD females and non-affected males. In addition to disease status, kidney mass adjusted for body weight (KM/BW) and urea levels (U), which are both strongly correlated (r2 = 0.752,p< 0.0001, Fig.1 ), were employed as phenotypes related to kidney function in the genetic analysis. The kidney mass ratios fell into two distributions shown in Figure2 . There are 54 affected rats represented in the upper distribution and 45 non-affected rats in the lower distribution. The means and standard deviations for KM/BW of the affected and non-affected hybrids are 4.95 ± 0.94 and 2.16 ± 0.22 respectively. The urea levels (U) do not display the same bimodal distribution in the studied population (Fig.2 ). Histograms of U in both affected and non affected hybrids are shown in Figure3 . The means and standard deviations for U in the affected and non-affected rats are 48.72 ± 7.71 mg/dl and 36.89 ± 2.69 mg/dl respectively.
The results from the genetic linkage study demonstrate the localisation of a gene responsible for PKD in Han:SPRD cy/+ near the marker D5Mit10 on rat chromosome 5. Similar results based on a genetic mapping of (BNx Han:SPRD cy/+) F2 rats have been described (18 ).
The disease locus is a strong determinant of kidney mass and plasma urea levels in the backcross. In PKD, cyst formation leads to fluid accumulation and a gain in kidney mass (defined as wet weight of both kidneys) in animals at the age examined in our study. Therefore the rise in kidney mass is an epiphenomenon of the PKD trait. Similar results could have been obtained by performing more detailed, but time-consuming, histo-morphological analysis of the kidneys. Plasma urea concentration is usually regarded as a good parameter of renal function in the rat, but differences between PKD affected and unaffected animals may be small in the early stage of the disease. Moreover, in recently weaned rats, plasma urea concentrations may also reflect nutritional protein intake or even protein catabolism due to an inadequate intake. Thus it is not surprising that, in comparison to kidney mass, a smaller proportion of the variance of plasma urea concentration is attributed to the PKD locus. It is likely that the correlation between the locus and plasma urea concentration would have been greater if older rats had been analysed. However, the correlation with kidney mass could well be lower in older animals, as kidney mass first decreases due to fibrosis and then increases again with age.
The gene responsible for PKD in this current rat model is neither PKD1 nor PKD2; therefore, we propose that this new locus be denoted PKDr1. The comparative map of the region of interest in rat, mouse and human, shown in Figure6 , has been established according to Bihoreauet al. (16 ), DeBryet al. (19 ), Dietrichet al. (20 ) and Mocket al. (21 ). Detailed linkage mapping of rat chromosome 5 places the PKD locus about 25 cM from the proenkephalin (PENK) gene. The homology region is likely to reside on human chromosome 8. However, according to the comparative map between mouse and human, it appears that the region between PENK and CYP4A contains genes localised on human chromosomes 6q and 9p. A more informative comparative map, and ultimately identification of PKDr1 through classical positional cloning techniques, will provide information for the study of non-PKD1 and non-PDK2 forms of the disease in humans, and new insights into its genetic causes.
Homozygous unaffected and heterozygous affected Han:SPRD rats exhibiting PKD were obtained from Dr Deerberg (Central Institute for Laboratory Animal Breeding, Hanover, Germany) (22 ) and highly inbred WOK (Wistar-Ottawa-Karlsburg) rats from the Diabetes Research Center in Karlsburg, Germany (23 ). The PKD rats were transferred into our laboratory when inbred to the 11th generation, while the WOK rats were already inbred up to the 21st generation. The PKD rats were then inbred up to the 19th generation before starting this experiment.
One affected male and one female PKD rat was crossed with WOK rats in order to obtain (PKD * WOK) F1 hybrids from which 18 male and 8 female F1 hybrids were backcrossed to respective WOK mate. The male rats derived from this [(PKD * WOK)F1 * WOK] first backcross were sacrificed when they had reached 2 months. During the study period all animals had free access to tap water and standard rat chow containing 19% protein (Altromin 1934®, Lage, Germany). The light cycle was 12 h, humidity 55% and room temperature 20oC.
At the time of sacrifice, the animals were bled from the aorta, the kidneys were removed and, after obtaining wet weight, both kidneys were fixed in 3% buffered formaldehyde and embedded in paraffin. The liver and spleen were snap frozen in liquid nitrogen and stored at -80oC for extraction of high-molecular-weight DNA.
For light microscopy, paraffin sections were stained with Hematoxylin-Eosin. The carrier status of each animal was established by histology. An animal was defined as affected, if in several visual fields at least occasionally small and/or medium-sized cysts up to the diameter of two glomeruli were noted. The histological examination was performed by two independent observers in a blinded fashion. In all cases agreement on the carrier status was obtained.
From the blood samples, serum urea concentration was determined by standard laboratory methods.
Genotyping was performed by PCR on the PKD*WOK backcross population (n = 91). The reaction volume was 20 µl containing 50 ng of genomic DNA, 0.25-0.75 µM of each primer, 45 mM Tris (pH 8.8), 11 mM (NH4)2SO4 (pH 8.8), 1.5 mM MgCl2, 6.7 mM [beta]-mercaptoethanol, 4.5 µM EDTA, 65.2 µM each dATP, dCTP, dGTP, dTTP and 0.4 UTaqDNA polymerase (Perkin Elmer). Thermocycling was initiated by 4 min of denaturation and followed by a touch-down protocol from 60-55oC or 55-50oC. PCR products were separated by electrophoresis on standard denaturing polyacrylamide gels and transferred to positive-charged nylon membrane (Pall). The membranes were hybridised with a primer labelled with [[alpha]-32P]dCTP using terminal transferase (Boehringer). Genotypes were determined after autoradiography.
Single factor segregation ratios were calculated for each marker in a linkage group before analysing for linkage and the genotypes at loci exhibiting significant distortion were checked for scoring errors. Subsequently, an initial linkage analysis was performed. The map obtained was used to order the file of marker loci so that double-recombinants could be identified in the data and the genotypes were checked for errors. The above tests and linkage analysis were performed using JoinMap v2.0 (24 ,25 ). A map of chromosome 5 markers in the region of linkage to PKD was constructed with the GMS program (26 ). Linkage to the PKD phenotype was evaluated initially by a contingency table analysis comparing the frequency of heterozygotes and homozygotes in backcross animals with and without disease. Multilocus linkage analysis was undertaken with the LINKAGE programs (27 ) assuming a completely penetrant, dominant disease model.
The authors are indebted to S. Meisinger, P. Prochazka, J. Christophel, I. Matera, M. Scaranari, M. Ferrari and D. Faruque for their technical assistance when performing the study. This work was supported by a grant from `Forschungsfonds der Fakultät für Klinische Medizin Mannheim der Universität Heidelberg', the Italian Telethon (grant D35), the EU (contract GENE-CT93-0040) and the Wellcome Trust Centre for Human Genetics.
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