Human Molecular Genetics, 2002, Vol. 11, No. 18 2165-2173
© 2002 Oxford University Press
Characterization of a major modifier locus for polycystic kidney disease (Modpkdr1) in the Han:SPRD(cy/+) rat in a region conserved with a mouse modifier locus for Alport syndrome
1The Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford OX3 7BN, UK, 2Medical Research Centre, Klinikum Mannheim, University of Heidelberg, D-68167 Mannheim, Germany and 3Institute of Veterinary Pathology, Free University of Berlin, D-14163 Berlin, Germany
Received May 9, 2002; Accepted June 28, 2002
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The genetic analysis of rodent disease models provides a powerful tool to investigate how modifier loci cause variation in the phenotypic expression of a disease. In order to test the existence of modifier loci influencing polycystic kidney disease (PKD) phenotypes, we derived a backcross between PKD susceptible Han:SPRD(cy/+) and control Brown Norway (BN) rats, and performed a whole-genome scan in 182 PKD affected hybrids showing different grades of disease severity. The genetic dissection of PKD in the cross allowed us to detect a modifier locus, Modpkdr1, on rat chromosome 8 that controls PKD severity, kidney mass and plasma urea concentration. Results from database searches and computational analyses demonstrated that the Modpkdr1 locus shows strong evidence of synteny conservation with human and mouse chromosomal regions controlling kidney diseases, including disease progression of Alport syndrome. Comparative genome mapping also provided an inventory of potential candidate genes for modifier(s) of PKD. Analyses of the coding regions for four strong candidates (Ctsh, Bcl2a1, Trpc1 and Slc21a2) in (cy/+), BN and Lewis rat strains did not reveal sequence variants that could be associated with PKD. The characterization of Modpkdr1 may provide new insights into modulating mechanisms involved in the pathogenesis of PKD that could delay disease progression in humans. It may also have strong implications in the identification of pathophysiological factors common to different renal disorders.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Autosomal dominant polycystic kidney disease (ADPKD) is the most common inherited renal disease, with a prevalence of 1 : 1000. A large proportion of affected patients reach end-stage renal disease by the age of 60 years. As a result, 5–10% of all patients on renal replacement therapy (i.e. hemodialysis, peritoneal dialysis or transplantation) suffer from polycystic kidney disease (PKD). Bilateral renal fluid-filled cysts are the main feature of ADPKD, but extrarenal manifestations are also common (1).
Human ADPKD is a genetically heterogeneous disease. Mutations in the PKD1 gene, mapped to chromosome 16p13.3 (2), are responsible for 85–90% of all cases of ADPKD. Mutations in the PKD2 gene, located on chromosome 4 (3), cause the disease in most of the remaining patients. In a small number of families, PKD segregates independently of either PKD1 or PKD2, which implies the involvement of at least a third, yet-to-be-characterized, gene (4,5). Another remarkable feature of this disease entity in humans is the broad phenotypic variability of the disease, even within the same family. The mechanisms underlying the variable severity of ADPKD may involve random somatic mutations in the kidney (6). In addition, predisposing genetic factors may influence the rate of somatic mutations or modulate the expression of the disease gene(s), and consequently control disease progression and/or severity (7). For obvious reasons, the identification of such modifier genes, which could have important therapeutic implications in ADPKD, is a virtually insurmountable problem in humans.
Owing to the complexity of genetic investigations of ADPKD in humans, rodent models that mimic the main pathophysiological features of the human disease have been used to investigate the genetic components controlling phenotypes relevant to PKD. The available mouse models generally reproduce the recessive form of PKD. In contrast, physiological and morphological phenotypes of the Han:SPRD(cy/+) rat strain closely resemble human ADPKD (8,9). In two experimental crosses independently derived from the Han:SPRD(cy/+) rat and either the resistant Wistar (WOK) (10) or Brown Norway (BN) (11) rats, PKD cosegregates with the locus Pkdr1 mapped to rat chromosome 5, which is not homologous to the human PKD1 and PKD2 genes (10). Although PKD in rodent models is inherited as a simple Mendelian trait, investigations in most mouse models have demonstrated that variability in disease expression is strongly influenced by as-yet unidentified modifier genes (12–18).
The aim of the present study was to test a possible analogy between mouse and rat models in the genetic modulation of PKD expression. We investigated the existence of modifier loci influencing PKD severity and related quantitative renal phenotypes in the Han:SPRD(cy/+) rat, and identified, on rat chromosome 8, a major quantitative trait locus (Modpkdr1) associated with disease severity. A high-resolution comparative genome analysis for this locus revealed potential candidate genes for ADPKD modifiers and strong homology relationships with known susceptibility loci for renal diseases in mouse and human.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Phenotypes
Of the 742 BC1 rats (386 males and 356 females;
2 test: P=0.271) derived from F1 parents exhibiting PKD, 372 displayed PKD of grade 1–5 (Fig. 1; see Materials and Methods) and 370 did not show evidence of PKD (2 test: P=0.941). This ratio of affected and unaffected animals in a backcross population is consistent with the influence of a single locus on PKD status. PKD was evenly distributed with gender (Fisher's exact test: P=0.164). Also, the mode of inheritance (maternal or paternal transmission) had no influence on the distribution of PKD in the BC1 generation (Fisher's exact test: P=0.767).
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Disease severity was more pronounced in this [Han: SPRD(cy/+)xBN]xBN backcross than in a previously described experimental cross derived from Han:SPRD(cy/+) and Wistar rats (10). Therefore an additional severity grade was added to our scoring system (Fig. 1E). In the present backcross study, we could not detect any impact of the line of transmission (maternal or paternal) on disease severity (Fisher's exact test: P=0.122).
The phenotypic characteristics of a subgroup of 182 BC1 rats selected for the genetic analysis of modifier loci are given in Table 1. Increased kidney mass corrected for body weight (kidney mass index, KM/BW) was significantly associated with high grades of PKD severity (Spearman correlation coefficient r=0.83354, P<0.0001). A similar trend was observed for serum urea (Spearman r=0.48580, P<0.0001) and to a lesser degree for serum creatinine (Spearman r=0.22643, P= 0.0022). KM/BW was highly correlated with urea concentration (Pearson correlation coefficient r=0.79608, P<0.0001) and to a lesser extent with creatinine (Pearson r=0.49975, P<0.0001). The kidney mass ratios fell into a broad distribution as shown in Figure 2, suggesting the involvement of modifier loci in the genetic control of this trait.
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Genetic mapping of the locus Pkdr1
Using genotypes generated with marker D5Mit9 in 182 affected and 136 non-affected hybrids of the [BNxHan:SPRD (cy/+)]xBN cross, we were able to confirm the existence of the locus Pkdr1 on rat chromosome 5 (
2 test: P=10-48) (10,11).
Linkage mapping of modifier loci for PKD
A genetic map was constructed using genotype data generated from the 212 markers typed in the selection of 182 PKD-affected hybrids. The markers spanned a total distance of 1701 cM (Kosambi), which is consistent with previously published linkage maps of the rat (19–22). The average distance between adjacent loci was 8.9 cM, with the largest gap (56 cM) on chromosome 20, and marker order was conserved with published maps (19–23).
Strongest evidence of genetic linkage was found between markers in a broad region of rat chromosome 8 and both PKD grades and kidney mass index (Fig. 3). The region of significant linkage with PKD grades spanned >30 cM between markers D8Got119 and D8Rat10. The strongest evidence of genetic linkage with PKD grades and kidney mass index was found at marker locus D8Rat17 (F=9.834, P=10-10 and F=6.552, P=2.8x10-7, respectively). This marker also showed evidence of significant linkage to serum urea concentration, whereas linkage to serum creatinine concentration was marginal and shifted towards marker D8Rat103 (data not shown). The quantitative trait locus (QTL) controlling kidney mass was responsible for an estimated 16% of the trait variance.
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Mean values of kidney traits were calculated for Han: SPRD(cy/+)/BN heterozygotes and BN/BN homozygotes at the Modpkdr1 locus. The Han:SPRD(cy/+) allele at the locus contributed to a significantly higher PKD grade and significantly higher kidney mass index values (Table 2). It was also associated with high levels of serum urea and creatinine, although the effect on creatinine was marginally significant.
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Marginal linkage was detected between PKD grading and/or KM/BW and marker loci in chromosomes 1, 2, 3 and 10 (data not shown).
Chromosomal mapping of new gene markers and comparative genome analysis of the locus Modpkdr1
Initial comparative mapping analysis was based on the localization of only two known genes (Col12a1 and Slc21a2) at the Modpkdr1 locus (22,23), whose homologues in mouse chromosome 9 and human chromosome 6q14.1 (COL12A1) and 3q22.1 (SLC21A2) allowed us to carry out extensive database searches and sequence homology analyses (Fig. 3). In addition, sequence homology searches using rat expressed sequence tags (ESTs) mapped to the Modpkdr1 locus (http://ratest.uiowa.edu/mapping-new/) identified a third conserved region on human chromosome 15q25.1 that was previously not reported but confirmed extensive synteny conservation between mouse chromosome 9 and rat chromosome 8 (Fig. 3).
Through database searches and computational analyses, we identified a total of 93 human gene sequences mapped in regions of chromosome 6q14.1–q15, 15q25.1 and 3q22.1–q24 likely to be homologous to rat chromosome 8. Detailed comparative mapping analysis of Modpkdr1 is available in our database (http://www.well.ox.ac.uk/rat_mapping_resources). A total of 70 corresponding rat gene and EST sequences were available in the GenBank database, whilst we were unable to identify rat orthologues for 23 human genes. Thirty-eight rat sequences were already localized in chromosome 8 by radiation hybrid typing, linkage or FISH analysis. We subsequently designed PCR primer pairs in 25 unassigned rat genes in order to localize them in our radiation hybrid map of the rat (24) and to refine homologies between human, rat and mouse genomes in the region of Modpkdr1. Fifteen gene markers that were not supported by our two-point analysis criterion (22) could not be mapped. The remaining 10 gene markers were assigned to the Modpkdr1 locus, and confirmed the expected synteny conservation between the three genomes at the locus. Information on primer sequences and PCR conditions for these markers, as well as map location and comparative genome analysis, are publicly available in our database (http://www.well.ox.ac.uk/rat_mapping_resources).
Sequence analysis of candidate genes
The candidate genes that we investigated were selected on the basis of their potential involvement in cyst formation. The coding sequence (CDS) of Ctsh was identical in Han:SPRD(cy/+), BN and LEW strains. Sequence alignment of Bcl2a1 CDS revealed only one single-nucleotide polymorphism (SNP), 325C
G in the LEW strain that leads to the replacement of a glycine by an alanine in the protein. In Trpc1 CDS, we detected one SNP, 1961GA in the BN strain. Two SNPs were characterized in Slc21a1: 719AT again in the LEW strain and 740AG specific to the Han:SPRD(cy/+) rat. All three substitutions are silent mutations. However, the variant 740AG in Slc21a1 may affect the secondary structure of the mRNA or its translation efficiency, and consequently the expression profile of the gene in the Han:SPRD(cy/+) rat.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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We have identified a major modifier locus, Modpkdr1, involved in the control of the number and size of cysts formed in the kidney of the Han:SPRD(cy/+) rat model of human ADPKD. In this strain, the presence of kidney cyst lesions is under the control of a single locus (Pkdr1) localized on rat chromosome 5 (10,11). We have now demonstrated that at least one genetic locus mapped to chromosome 8 strongly influences disease severity in an experimental cross derived from Han:SPRD (cy/+) and BN rats, and modulates the effects of mutation(s) in gene(s) at the locus Pkdr1 on PKD phenotypes. Although genetic heterogeneity of ADPKD can explain part of the phenotypic variability in humans, it does not account for clinical variability observed in intrafamilial genetic studies. It is well accepted that, in addition to germline mutations in either of the human PKD genes, genetic factors such as modifiers may affect the disease expression (7). The detection of such modifier genes in PKD patients is complicated by the involvement of environmental factors in disease progression and by the experimental design of genetic studies that has to be applied in order to address the concept of modifier loci. These difficulties can be minimized in animal models for ADPKD that allow extensive experimental control of both genetic and environmental variables. The effect of modifier loci in animal models of human diseases results in differential phenotypic effects of mutations when analyzed in the context of different genetic backgrounds (25). As the involvement of environmental factors can be theoretically ruled out in inbred animal models, these phenotype alterations are usually attributed to expression of modifier alleles that are strain-specific and/or can be uncovered only in an experimental cross produced from a specific pair of affected and unaffected strains.
Results from the phenotype analysis of PKD variables in experimental crosses derived from the Han:SPRD(cy/+) rat demonstrate that disease severity is more pronounced in [Han:SPRD(cy/+)xBN]xBN progenies than in [Han: SPRD(cy/+)xWOK]xWOK hybrids maintained under identical environmental conditions (10,26). These observations suggest that genetic factors other than Pkdr1 mutation(s) exacerbate the PKD phenotypes when expressed in a BN genetic background. Significant strain differences in the expression of PKD phenotypes have also been described in PKD mouse models. For example, PKD due to mutations in the pcy gene is more severe in a DBA/2 background than in a C57BL/6 background (27). Approximately 30% of [DBA/2 pcyxCAST]F2 affected hybrids showed an increased severity and accelerated progression of the PKD disease when compared with the parental strain DBA/2(pcy/pcy) (13).
Subsequent genetic analysis in experimental crosses produced from various mouse models of recessive PKD has led to the identification of different strong or putative modifier loci on mouse chromosomes 1, 4, 10, 16 and 19, suggesting that multiple genes are involved in renal cystogenesis and that epistatic interactions between them are possible (7,16). The characterization of the PKD modifier locus Modpkdr1 in the Han:SPRD(cy/+) rat further emphasizes the importance and complexity of genetic factors influencing disease expression in the context of a dominant form of the disease. Evidence of significant genetic linkage with PKD grades and kidney function phenotypes was found in a broad region of rat chromosome 8. We have demonstrated that Han:SPRD(cy/+) alleles at the Modpkdr1 locus contribute significantly to the expression of severe PKD traits. Conversely, our results suggest that more permissive BN alleles at this locus may at least partly account for increased PKD grades in the cross.
Comparative genome analysis between rat, mouse and human represents a powerful approach for defining homology relationships between a rat susceptibility locus and mouse/human chromosomal regions that may already be characterized for related susceptibility loci or can be considered as candidates for linkage and/or association studies. Evidence of synteny conservation between rodent and human disease susceptibility loci has already been observed. For example, a modifier locus for cystic fibrosis mapped to chromosome 7 in the Cftrm1HSC mouse was replicated in the syntenic region of human chromosome 19q13.2–q13.4 (28). Until the sequence of the mouse, rat and human genomes is completed and fully annotated for genes, comparative mapping analysis is based on the comparison of localization of known genes in these species (20,23). The most recent studies have demonstrated extensive synteny conservation between rat and mouse chromosomes, whereas homology relationships between rat and human genomes remain partly defined (20,23). Following computational analyses and RH mapping, we have shown that the locus Modpkdr1 shows evidence of synteny conservation with mouse chromosome 9 and is homologous to regions of human chromosomes 6q14–q15, 15q25.1 and 3q22.1–q24.
The region of mouse chromosome 9 directly homologous to Modpkdr1 contains genes involved in the progression of renal disease in the Col4a3-/- mouse model of Alport syndrome, a hereditary glomerular disorder (29). Both this disease and ADPKD share common pathophysiological features that may be influenced by the same modifier gene or closely related modifier genes. Both Col4a3-/- mice and Han:SPRD(cy/+) rats display thickened basement membrane (BM) at an early stage of the disease, and tubulointerstitial fibrosis and inflammation at ESRF (8,29). Accumulation of the 
1 chain of type IV collagen in the areas of BM deterioration is observed in both ADPKD and Alport syndrome (29,30). Alterations in expression of BM components and interstitial collagens associated with cyst development have also been reported in DBA/2FG–pcy and cpk mouse models of PKD (31,32). Therefore, the regulation of collagen 1 (IV) expression may influence disease progression in two different renal pathologies.
The region between marker loci Slc21a2 and D8Rat10 on rat chromosome 8 that remains significantly linked to PKD grades shows evidence of synteny conservation with the mouse pcy and the human NPHP3 loci, both of which are involved in PKD phenotypes (ADPKD and adolescent nephronophthisis, respectively) (33), and are characterized by thickened tubular BM and marked interstitial fibrosis (34,35). Therefore, we cannot rule out the possibility that genetic variants in the rat gene corresponding to mouse pcy and/or human NPHP3 may influence PKD severity in the Han:SPRD(cy/+) rat model. However, at this stage of the genetic analysis of Modpkdr1, it is not possible to determine whether pcy and/or NPHP3 are candidate genes for Modpkdr1, or if they represent independent loci also involved in the control of PKD severity in the Han:SPRD(cy/+) rat. The hypothesis of several genetic alterations in the same chromosomal region participating in the QTL effect derives from genetic studies in rodent models of essential hypertension and type 1 diabetes (36,37). For example, the construction and characterization of rat congenic lines for high-blood-pressure susceptibility loci have shown that a QTL detected in an experimental cross is likely to be the result of the effects of genetic variants in several independent genes at the locus (36). Further analyses of the region of rat chromosome 8, for example in congenic lines of the Han:SPRD(cy/+) rat, will allow us to test the relevance of the modifier locus for Alport syndrome and the loci of pcy and NPHP3 to Modpkdr1.
The computational analysis that we carried out using gene maps and genomic sequences of human and mouse chromosomal regions conserved with the Modpkdr1 locus provided us with an inventory of potential candidate genes for PKD modifiers. Owing to the size of this QTL, we prioritized our investigations to strong candidate genes that may be involved in cyst formation resulting from cell proliferation, fluid accumulation and/or increased degradation of the extracellular matrix (7) that promotes matrix deposition and fibrosis as observed in Han:SPRD(cy/+) rat kidneys (38). Among the known candidates mapped to the locus Modpkdr1, we analysed Ctsh (cathepsin H), Bcl2a1 (Bcl2-related protein A1), Trpc1 (transient receptor potential channel 1) and Slc21a2 (prostaglandin transporter). In addition, Adamts7 (encoding a disintegrin-like and metalloproteinase with thrombospondin type 1 motif 7), which maps to a region of mouse chromosome 9 conserved with Modpkdr1, is not sequenced in the rat and still unmapped in this species, and could not be investigated.
CTSH is a cysteine-dependent aminopeptidase, which plays an important role in regulating intracellular protein degradation and turnover. A previous study showed that Ctsh proteinase activity and levels were altered in proximal tubules from 2-month-old Han:SPRD(cy/+) rats, whereas mRNA levels were unchanged when compared with healthy animals (39). Variants in Ctsh that reduce its activity would increase metalloproteinase accumulation and extracellular matrix degradation. ADAMTS7, a member of a membrane-anchored glycoprotein family (40), may also degrade extracellular matrix components through its metalloproteinase activity, and inhibit binding of cells to the matrix through its disintegrin domain. BCL2A1 has similar antiapoptotic activity to BCL2, preventing oxidative damage to cellular constituents. Increased apoptosis is also a key feature of ADPKD. It has been detected in kidneys of newborn Han:SPRD(cy/cy) and 2-week-old (cy/+) rats, and correlates with changes in expression of Bcl2 family proteins (41). Of additional interest are the facts that the locus harbouring Bcl2 has been identified as a modifier locus of PKD in kat2j (17) and jck (12) mice and that Bcl2-deficient mice display severe PKD characterized by dilated proximal and distal tubular segments and hyperproliferation of epithelium and interstitium (42). TRPC1 appears to encode a channel that allows calcium influx in response to depletion of intracellular calcium pools (store-operated calcium entry) (43). It has been shown that PKD2 can specifically interact with TRPC1, and this interaction contributes to the functional role of PKD2 in the maintenance of renal epithelial architecture, thus eliminating the possibility of non-specific interactions between PKD2 and other homologous channels (44). SLC21A2 is one of the prostaglandin transporters that mediate the efflux of newly synthesized prostaglandins from cells, their epithelial transport, and their clearance and degradation. Changes in prostaglandin levels are known to modify renal ion transport in part through cAMP production. It has been suggested that cAMP increased by prostaglandin E1 (PGE1) can induce in vitro cyst formation in Madin–Darby canine kidney cells (45). PGE2-induced cAMP can stimulate in vitro the proliferation of human ADPKD renal cyst epithelial cells, whereas it has no impact on the rate of proliferation of normal kidney cortex epithelial cells (46).
Although cDNA sequencing of Ctsh, Bcl2a1, Trpc1 and Slc21a2 failed to identify variants likely to alter gene function in Han:SPRD(cy/+) and BN rats, regulatory regions and intron splicing sites have not been screened for sequence variants. We cannot rule out the existence of mutations in non-coding sequences of these genes (as well as in other genes at the locus Modpkdr1) that would alter their expression and subsequently modify the phenotypic effect of the gene underlying Pkdr1.
The characterization of the Modpkdr1 locus and the role of genetic variants at the locus on PKD severity have important implications for our understanding of the modulation of disease severity and the strong clinical heterogeneity of ADPKD. Evidence of synteny conservation between Modpkdr1 and mouse/human loci involved in distinct renal disorders sharing similar features with PKD, including basement membrane structural modifications and interstitial fibrosis, may indicate the effect of a single gene or a group of linked genes on common morphological and/or physiological responses to different renal pathologies. The demonstration of modulations in renal disease phenotypes controlled by sequence variants in the same genes will have important therapeutic implications.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Animals
Homozygous unaffected and heterozygous affected Han:SPRD rats were initially obtained from Dr Deerberg (Central Institute for Laboratory Animal Breeding, Hannover, Germany) (10). The rats were obtained at the 11th generation of inbreeding, and a further inbreeding of 18 generations was performed in our laboratory (http://www.informatics.jax.org/external/festing/rat/docs/PKD.shtml). BN rats were obtained from Elevage Janvier, Le Geneste St Ille, France.
An initial F1 generation was produced by mating three F0 heterozygous Han:SPRD(cy/+) males with six control F0 BN females. A large cohort of 742 first-backcross rats (BC1) was derived from BN rats mated to either affected F1(cy/+) males (paternal line) or females (maternal line). A backcross was preferred to an intercross, because cy/cy mutants die within 28 days postnatal, before the detection of the disease can be clearly assessed in the Han:SPRD(cy/+) hybrids and its progression analysed. The BN rat strain was chosen for this study because (i) it does not develop evidence of ADPKD, (ii) the Pkdr1 locus was replicated in a Han:SPRD(cy/+)xBN cross (11) and (iii) a preliminary analysis of allele variation of microsatellites between rat strains (19) (http://www.well. ox.ac.uk/
bihoreau/polytab.html) showed that a polymorphism rate of 76%, appropriate for linkage mapping, is expected between Han:SPRD(cy/+) and BN rats.
During the study period, all animals had free access to tap water and standard rat chow containing 19% protein (ssniff, Soest, Lage, Germany). The light cycle was 12 hours, humidity 55% and room temperature 20°C. All animals of the backcross generation were sacrificed at day 36. On the day of sacrifice, body weight was determined, blood samples were collected from the orbital plexus under ketamin/xylazine anaesthesia (100 mg/5 mg/kg body weight), and kidneys were removed and fixed in 3% buffered formaldehyde after wet weight had been determined. The kidneys were then embedded in paraffin for further structural analyses.
Phenotype analysis
The disease carrier status of each animal of the backcross population was established by light-microscopic analysis of kidney sections stained with hematoxylin–eosin. Animals were defined as PKD-affected when small and/or medium-sized cysts up to the diameter of a glomerulum were observed at least occasionally in several visual fields. The histological examination was performed by two independent observers in a blinded fashion. In all cases, agreement on the carrier status was obtained. Presence and absence of renal cysts was used only for replication of the Pkdr1 locus in the cross. The extent of cyst formation was assessed using a previously described scoring system (26). Furthermore, the grading system was validated following morphometric analysis in order to determine both the average of total cyst number (CN) and percentage of cyst area of the total kidney area (%CA) for each grade:
Grade 1 (Fig. 1A): occasional small and/or medium-sized cysts with a diameter of up to two glomeruli, with cysts only in a few visual fields (CN=31.6±7.8;%CA=1.0±0.3).
Grade 2 (Fig. 1B): few small and/or up to five medium-sized cysts per visual field, rarely large cysts exhibiting a diameter larger than two glomeruli, with cysts not yet in every visual field (CN=52.8±20.9;%CA=1.5±0.6).
Grade 3 (Fig. 1C): several small and/or up to 10 medium-sized cysts and a few large cysts, with cysts in every visual field (CN=84.2±19.8;%CA=2.6±0.5).
Grade 4 (Fig. 1D): a large number of small or medium-sized and/or at least two large cysts per visual field, with cysts in every visual field and the occurrence of up to three network-like structures (CN=124.8±44.3;%CA=3.5±0.8).
Grade 5 (Fig. 1E): practically no normal kidney tissue visible and histology exhibiting only large cysts and network-like structures similar to that seen in homozygous Han:SPRD (cy/cy) rats (8) (CN=268.7±50.8;%CA=5.45±0.8).
Serum urea and creatinine were determined by standard laboratory methods (Hitachi 911 Autoanalyser, Roche Diagnostics, Mannheim, Germany).
Genotype analysis
A subset of 318 affected and non-affected backcross hybrids were used for the genetic analysis of the locus Pkdr1, whereas a subgroup of 182 backcross hybrids exhibiting various PKD grades and similarly affected on both kidneys were selected for the chromosomal mapping of PKD modifier loci.
The microsatellite marker D5Mit9 mapped in the close vicinity of the locus Pkdr1 (10,11) was chosen to test the cosegregation of (cy/+) genotypes at the locus with the presence of PKD in the cross. For the genome-wide search of PKD modifier loci, a total of 212 microsatellite markers exhibiting allele variations between the Han:SPRD(cy/+) and BN rat strains were selected in our database (http://www.well. ox.ac.uk/rat_mapping_resources) and used to determine the genotypes of the subset of 182 affected backcross hybrids. All selected markers have already been genetically mapped in various rat crosses (19–22) and/or localized in the rat genome using the T55 rat radiation hybrid panel (23). Markers used in this study were chosen to cover the 21 chromosomes of the rat genome, with an average spacing of 10 cM between loci. Genotypes were determined following PCR amplification and gel electrophoresis of PCR products as previously described (10). Oligonucleotides were synthesized commercially by Genosys Biotechnologies (Pampisford, UK).
Construction of the genetic maps
Prior to linkage analysis, JoinMap version 2.0 (47) was used to calculate single-factor segregation ratios for each marker in a linkage group. The genotypes were checked for all individuals at markers that were exhibiting significant distorted segregation. Subsequently, initial maps were created and double recombination events were identified with JoinMap, and the data corresponding to these were verified. The JoinMap module for genotype checking calculates for all loci and for all individuals the probability of obtaining the present genotype, conditional on both map distances and genotypes at the two flanking loci. We considered unexplained genotypes to be those having a threshold >3 for the test statistic of log10(1/P).
Statistics
Phenotypic data were analysed with the SAS System version 8.1 (SAS Institute Inc. Carry, NC). The following procedures were applied: PROC ANOVA (analysis of variance), PROC CORR (Pearson and Spearman correlation coefficients, as appropriate), PROC FREQ (Fisher's exact test for 2x2 or larger tables or 2 test) and PROC MEANS (mean and SD). All data are given as 
±SD. Whenever computational resources allowed (n<400), Fisher's exact test instead of the 2 test was applied.
Linkage analysis
Linkage between marker genotypes and kidney mass index was initially evaluated using the MAPMAKER/QTL computer package (48). Non-parametric linkage analysis for non-normalized traits, such as PKD grading, was performed by an ANOVA test followed by a permutation test (n=10 000) in order to evaluate the threshold of significance for each pair of genetic–phenotypic markers (49).
Comparative genome analysis for the locus Modpkdr1
In order to initially anchor rat, mouse and human gene maps at the locus Modpkdr1, localization of orthologues for two genes (Slc21a2 and Col12a1) already mapped in the region of linkage (20,23) was obtained by querying the mammalian homology databases (http://www.ncbi.nlm.nih.gov/Homology/ and http://www.informatics.jax.org/). A list of human genes mapped in the interval was established using both the Human Genome Browser (http://genome.ucsc.edu/) and Ensembl (http://www.ensembl.org/), which are sequence annotation systems of the working draft of the human genome, and Genemap (http://www.ncbi.nlm.nih.gov/genemap99), which is based on RH mapping of the human genome. Rat sequences (mRNAs and ESTs) homologous to each human sequence were accessed, where possible, via Locus Link (http://www.ncbi.nlm.mih.gov/LocusLink), which provides connections to various NCBI resources, including HomoloGene and Map Viewer. In addition, human sequences were used to search for rat orthologues (BLASTn) in EMBL rodent EST and sequence databases, ESTs, Unigene rat sequences and unfinished rat genomic sequence (http://hgsc.bcm.tmc.edu/projects/rat/). Conversely, each rat EST already mapped to Modpkdr1 was searched (BLASTn) for cDNA sequences and gene names in the TIGR rat gene index (http://www.tigr.org/tdb/rgi/ index.html) and REFSEQ (http://www.ncbi.nlm.mih.gov/LocusLink/refseq.html). BLASTX was used to search for functional similarity between potential orthologues.
Chromosomal mapping of gene loci in the rat RH map
Unmapped rat gene and EST sequences that showed evidence of homology with mouse/human genes assigned to regions conserved with Modpkdr1 were localized in the rat genome using the T55 rat radiation hybrid (RH) panel (24) (Research Genetics, Huntsville, AL). Oligonucleotides were designed in the rat sequences using the OLIGO 4.0 computer program (National Biosciences Inc., Plymouth, MN). Optimization of the PCR reactions, data analysis and integration of the rat gene markers in our RH framework map of the rat (24) were performed as previously described (22).
Sequence analysis of candidate genes in rat strains
Total RNA was prepared from kidneys of Han:SPRD(cy/+), BN and Lewis (LEW) rat strains using the TRIzol reagent (Life Technologies, Paisley, UK). cDNAs were synthesized for sequence analysis (SMART PCR cDNA synthesis kit, BD CLONTECH, Basingstoke, UK). PCR primer pairs, amplifying overlapping cDNA fragments, were designed along the full-length coding sequence for Ctsh (GenBank accession no. NM_012939), Bcl2a1 (GenBank accession no. AF378332), Trpc1 (GenBank accession no. AF0612266) and Scl21a2 (GenBank accession no. NM_022667) using the OLIGO 4.0 computer program, and PCR products were sequenced in both directions (BigDye Terminator kit, Perkin-Elmer/Applied Biosystems, Warrington, UK). Sequences derived from the rat strains were compared using the Align computer program of the DNAStar package (Madison, WI).
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ACKNOWLEDGEMENTS |
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The authors are indebted to J. Christophel for her technical assistance and P. Wilson and M. Rauscher for their help in preparing this manuscript. This work was supported by grants from the National Kidney Research Fund (Grant R27/2/99), the European Commission (Grant QLRT-2000-01104), the DAAD (Deutscher Akademischer Austauschdienst, ARC Project 313-ARC-XIV-00/30; 00/27685), the British Council (ARC Project 1125.00) and the Wellcome Trust. D.G. holds a Wellcome senior fellowship in basic biomedical science.
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FOOTNOTES |
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* To whom correspondence should be addressed. Tel: +44 1865287648; Fax: +44 1865287533; Email: bihoreau{at}well.ox.ac.uk
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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