Blood pressure QTL that differentiate Dahl salt-sensitive and spontaneously hypertensive rats

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1 Physiol Genomics 3: 33 38, Blood pressure QTL that differentiate Dahl salt-sensitive and spontaneously hypertensive rats MICHAEL R. GARRETT, YASSER SAAD, HOWARD DENE, AND JOHN P. RAPP Department of Physiology and Molecular Medicine, Medical College of Ohio, Toledo, Ohio Received 15 March 2000; accepted in final form 13 May 2000 Garrett, Michael R., Yasser Saad, Howard Dene, and John P. Rapp. Blood pressure QTL that differentiate Dahl salt-sensitive and spontaneously hypertensive rats. Physiol Genomics 3: 33 38, Our purpose was to define quantitative trait loci (QTL) for blood pressure that differ between two widely used hypertensive rat strains, the Dahl saltsensitive (S) rat and the spontaneously hypertensive rat (SHR). A genome scan was done on an F 2 (S SHR) population fed 8% NaCl for 4 wk. Three blood pressure QTL were detected, one on each of rat chromosomes (chr) 3, 8, and 9. For the chr 3 QTL the SHR allele increased blood pressure, and for chr 8 and 9 the S allele increased blood pressure. The QTL on chr 9 was exceptionally strong, having a LOD score of 7.3 and accounting for 30% of the phenotypic variance and a difference of 40 mmhg between homozygotes. A review of the literature in conjunction with the present data suggests that S and SHR are not different for the previously described prominent blood pressure QTL on chr 1, 2, 10, and 13. QTL for body weight on chr 4, 12, 18, and 20, each with an effect of about 30 g, were incidentally observed. high blood pressure; body weight; heart weight; SHR; Dahl S rats IN ESSENTIALLY ALL PREVIOUSLY published genome scans for linkage to blood pressure using genetically hypertensive rats, a hypertensive strain has been crossed with a normotensive strain. This has yielded a wealth of data with putative quantitative trait loci (QTL) for blood pressure being found on all rat chromosomes (chr) except chr 6, 11, and 15. The use of hypertensive rats in the genetic dissection of blood pressure has recently been reviewed (32). There are nine inbred strains of rats that have been selectively bred for high blood pressure. Of these nine strains, by far the most genetic analyses have been done with the Dahl salt-sensitive (S) rat and the spontaneously hypertensive rat (SHR) (32). Both strains develop progressive hypertension with growth, but of course the S rats were developed by selectively breeding for their blood pressure when fed a high-salt (NaCl) diet (7, 33), and SHR were not (29). A priori there is no reason to expect that the S and SHR are hypertensive Article published online before print. See web site for date of publication (). Address for reprint requests and other correspondence: J. P. Rapp, Dept. of Physiology and Molecular Medicine, Medical College of Ohio, 3035 Arlington Ave., Toledo, OH ( jrapp@mco.edu). for the same reasons, i.e., that they both carry alleles for increased blood pressure (plus alleles) at the same QTL. In fact because the S and SHR were developed from different outbred stocks (Sprague-Dawley and Wistar, respectively) and because S (but not SHR) were selected for blood pressure when fed a high-salt (NaCl) diet, one can reasonably expect that the two strains will carry contrasting alleles at some blood pressure QTL. The purpose of the present work is to define the blood pressure QTL that differ between S and SHR. A corollary is that this work also defines the major known blood pressure QTL where S and SHR do not carry contrasting alleles. MATERIALS AND METHODS Animal procedures. Dahl salt-sensitive rats (SS/Jr) were obtained from our colony and will be referred to as S. Spontaneously hypertensive rats (SHR/NHsd) were obtained from Harlan Sprague-Dawley (Indianapolis, IN) and will be referred to as SHR. An F 2 population of 108 male rats was produced by crossing S females to SHR males to yield F 1, and then intercrossing F 1 to make F 2.F 2 rats were weaned at 30 days of age and placed on a high-salt diet (8% NaCl Teklad diet TD 82050) at 37 days of age. After 4 wk on a high-salt diet, systolic blood pressure was measured. Systolic blood pressure was measured by the tail cuff method on ether-anesthetized rats warmed to 28 C (14). Blood pressure was measured on each rat on two separate days during a 5-day period with at least 2 days rest between measurements. During each session, three to four consistent reading were taken and averaged for that session. The final blood pressure for each rat was the average of the two session readings. The rats were subsequently killed by an overdose of pentobarbital sodium, and pieces of liver were frozen at 70 C for subsequent DNA extraction. Body weight and heart weight were measured. Adjusted heart weight was calculated by adjusting heart weight for differences in body weight by regression analysis (30) using programs from SPSS (Chicago, IL). The F 2 rats had been bred and their blood pressure studied in January 1989, but the genome scan on archived DNA was done in SHR male rats were also obtained from Harlan Sprague- Dawley in 1999 for comparison to S. Ten S and 10 SHR were fed low-salt diet (Teklad diet 7034; Harlan Teklad, Madison, WI) from weaning to 53 days of age at which point they were switched to 4% NaCl diet (Teklad diet 83033). Blood pressure was taken at week 0, week 2, and week 4 from the start of the 4% NaCl diet. Blood pressure was measured by the tail cuff method in the conscious restrained rat using semiautomated /00 $5.00 Copyright 2000 the American Physiological Society 33

2 34 GENOME SCAN FOR BLOOD PRESSURE COMPARING S AND SHR Table 1. Temporal comparison of blood pressure and body weight for S and SHR fed on 4% NaCl diet S SHR P Value (t-test) Age 53 days, 0.3% NaCl diet BP, mmhg Body wt, g Age 67 days, 2 wk on 4% NaCl diet BP, mmhg Body wt, g Age 81 days, 4 wk on 4% NaCl diet BP, mmhg Body wt, g Values are means SE. There were 10 rats in each group. S, Dahl salt-sensitive rat; SHR, spontaneously hypertensive rat. BP, blood pressure. equipment made by IITC Life Sciences Instruments (Woodland Hills, CA). Genotyping. Genomic DNA was prepared from the livers of F 2 rats by standard chloroform:phenol methodology (1). Genotyping was done using microsatellite markers amplified by the PCR and evaluated by electrophoresis as previously described (10). Marker genotypes were scored by two independent observers. Markers were selected from those developed by the following sources; 1) Massachusetts Institute of Technology (MIT, Cambridge, MA; wi.mit.edu), available from Research Genetics (Huntsville, AL); 2) Wellcome Trust Centre for Human Genomics (Oxford, UK; available from Genosys (Cambridge, UK); 3) University of Iowa (39); and 4) literature sources (8, 9, 12, 13, 16) (Medical College of Ohio; mco.edu/depts/physiology/research). Markers were spaced as evenly as possible to provide a marker every cm. Linkage and statistical analysis. Linkage analysis and QTL localization were performed with Mapmaker/EXP (22, 24) and Mapmaker/QTL (25, 31) programs. Linkage maps were constructed using the Kosambi mapping function to calculate distances between markers. Detection of an association between blood pressure, adjusted heart weight, or body weight with marker loci for QTL in the F 2 population was first carried out using an unconstrained model (free genetics) in Mapmaker/QTL using a subset of 92 randomly selected rats that were initially genotyped. Once suggestive evidence for association of phenotype with marker loci was established, markers in the region of a blood pressure QTL were genotyped on the remaining 16 rats and other genetic models were applied (dominant, recessive, and additive). Models that gave a LOD score more than 1 unit below the unconstrained model were ruled out. Markers were determined to be significant or suggestive with respect to linkage to phenotype using criteria as recommended by Lander and Kruglyak (23). Determination of the phenotypic effect for markers with significant or suggestive linkage to phenotype was done by selecting one marker nearest to the QTL peak and performing a one-way ANOVA using programs from SPSS. Interactions. Interactions throughout the genome were examined using software provided by Dr. Gary Churchill of the Jackson Laboratory (2). The method examined all pairs of marker loci for an interaction with a given phenotype. Permutation testing assessed the significance of pairwise effects. RESULTS Comparison of S vs. SHR. Table 1 gives the blood pressure data comparing S and SHR fed 4% NaCl diet. At 53 days of age on low-salt diet, S and SHR had similar blood pressure, 151 and 154 mmhg, respectively. On 4% NaCl diet the blood pressure of S increased more rapidly than SHR, so that S were 39 mmhg higher than SHR after 2 wk on the high-salt diet. This difference persisted at 4 wk on high-salt diet. SHR were considerably smaller than S at 53 days of age, and this difference persisted on the high-salt diet. Table 2. Genomic coverage for F 2 (S SHR) genome scan Chromosome Genetic Length, cm (Kosambi) % Length Linkage Map % Length Physical Map* Number of Markers Max Distance Between Adjacent Markers % Chromosome Within 10 cm of a Marker X Y 3.20 Total 1, % * From the Committee for Standardized Karyotype of Rattus norvegicus (6).

3 GENOME SCAN FOR BLOOD PRESSURE COMPARING S AND SHR 35 Genome coverage. The genomic coverage obtained from using 214 microsatellite markers on the F 2 (S SHR) population is given in Table 2. The lengths of linkage maps in Table 2 are based on genotype data collected on 92 randomly selected animals. The average spacing between markers was 8.1 cm, with an average of 99.7% of the genome covered within 10 cm of a marker (Table 2). Linkage to blood pressure, adjusted heart weight, and/or body weight. The results of the QTL analysis for blood pressure are summarized in Fig. 1. The LOD plots are given for chromosomes that yielded a LOD score at least at the suggestive level of significance for their respective genetic models. LOD plots for the adjusted heart weight and body weight have been omitted, but the linkage data are summarized in Table 3. Table 3 lists the marker that lies closest to the QTL peak, the variance explained by the QTL, and the phenotypic effect. The phenotypic effect is the average phenotype of rats homozygous for the S allele minus the average phenotype of rats homozygous for the SHR allele (Table 3). The average values for each phenotype by genotype are listed in Table 4, along with the results of one-way ANOVA for the marker locus nearest the LOD peak. As shown in Fig. 1, QTL for blood pressure were localized to chromosomes (chr) 3, 8, and 9. Two of the QTL (chr 3 and 8) only attained LOD scores that are considered suggestive for linkage to blood pressure. Chromosome 9 was the only chromosome that yielded a LOD score that clearly can be considered significant for linkage to blood pressure. It had a maximum LOD score of 7.3 and explained 30% of the total variance in blood pressure in the F 2 (S SHR) population. For the blood pressure QTL located on chr 3, the S allele acted to lower blood pressure and the SHR allele increased blood pressure. For the QTL located on chr 8 and 9, the opposite was the case; the S alleles increased blood pressure, and the SHR alleles lowered it (Tables 3 and 4). Figure 2 shows the relationship between adjusted heart weight and blood pressure (r 0.79, P ). Significant linkage to adjusted heart weight using LOD plots and the criteria of Lander and Kruglyak (23) was only found on chr 9 and was localized to the same region as the blood pressure peak. The direction of the adjusted heart weight change on chr 9 (Tables 3 and 4) was consistent with the blood pressure change and the positive relationship between adjusted heart weight and blood pressure. The adjusted heart weight yielded a suggestive QTL on chr 3 in the same position as the blood pressure QTL, and the direction of change of adjusted heart weight was consistent with the blood pressure changes (Tables 3 and 4). In Table 3 we also Fig. 1. LOD plots for linkage of blood pressure to rat chromosomes (chr) 3 (top), 8 (middle), and 9 (bottom) for an F 2 (S SHR) population fed 8% NaCl for 4 wk. The dashed line in each plot represents the LOD threshold for suggestive significance, and the solid line is the threshold for significance (23). For the chr 9 plot the heavy vertical bars to the right of the genetic map represent the 1-LOD intervals, and the extensions on these bars are the 2-LOD intervals for two experiments where a quantitative trait locus (QTL) was found on chr 9. The bar labeled S vs. SHR is for the present data derived from the LOD plot shown. The bar labeled S vs. R is the for a blood pressure QTL described on chr 9 in an F 2 population derived from S and Dahl salt-resistant (R) rats (36).

4 36 GENOME SCAN FOR BLOOD PRESSURE COMPARING S AND SHR Table 3. Putative QTL for blood pressure, adjusted heart weight, and body weight in the F 2 (S SHR) genome scan Phenotype Chr Marker Model Max LOD % Variance Explained Phenotypic Effect SBP, mmhg 3 D3Rat53 Additive D8Rat24 Dominant D9Rat26 Additive Adjusted heart wt, mg 3 D3Rat53 Additive D9Rat26 Additive Body wt, g 4 D4Rat122 Recessive D12Rat57 Recessive D18Mgh3 Additive D20Rat31 Recessive Under the Model column, recessive indicates that SHR was recessive to S, and dominant indicates that SHR was dominant to S. Phenotypic effect is the average phenotype of rats homozygous for the S allele minus the average phenotype of rats homozygous for the SHR allele. A positive value indicates that the S rats carry the plus allele, and a negative value indicates that the S rats carry the minus allele when compared with SHR. Chr, chromosome. QTL, quatitative trait loci; and SBP, systolic blood pressure. list the data for the one-way ANOVA for adjusted heart weight associated with the blood pressure QTL on chr 8. In contrast to chr 3 and 9, the adjusted heart weight data for chr 8 was not significant despite a substantial effect of the chr 8 QTL on blood pressure (Tables 3 and 4). A small change in adjusted heart weight associated with the blood pressure QTL on chr 8, although not significant, is in the expected direction. In addition to blood pressure and heart weight QTL, several QTL for body weight were found on chr 4, 12, 18, and 20. All these chromosomes, however, yielded LOD scores that provided only suggestive evidence for linkage to body weight (Tables 3 and 4). Interactions were sought by pairwise comparisons of every marker with every other marker in the genome scan. This was done for blood pressure, adjusted heart weight, and body weight, but no interactions were found. DISCUSSION The F 2 (S SHR) population reported here had been produced in 1989 and studied for cosegregation of blood pressure with the renin locus (34). Those data were not particularly informative, but the genome scan completed in 1999 offers several interesting features. Because two hypertensive strains were crossed, one expects the genetic background to be permissive for development of hypertension. This was certainly the case, because as reported earlier (34) the population had to be studied after only 4 wk on 8% NaCl diet to avoid death of the rats with the highest blood pressure. In several other F 2 populations, where S were crossed with a normotensive strain, the most responsive rats in the populations reached the lethal stage of hypertension only after 8 12 wk on 8% NaCl diet (34). Of the three putative blood pressure QTL reported here, all have been observed previously in various crosses of S or SHR with normotensive strains. The blood pressure QTL on the proximal end of chr 3 supports the data of Cicila et al. (3) showing two blood pressure QTL on chr 3, one on the proximal end and one on the distal end, in crosses involving S and R rats. The QTL found here is on the proximal end and is probably the same one localized there by Cicila et al. (3) and also observed in the genome scan of an F 2 (S Lewis) population (15). Blood pressure QTL on chr 3 have also been reported in crosses involving strokeprone SHR and Wistar-Kyoto rats (5, 26), but because of poor localization of the QTL to roughly the central region of chr 3, as well as major discrepancies in Table 4. Average phenotypic effect by genotype for blood pressure, adjusted heart weight, and body weight in the F 2 (S SHR) genome scan Phenotype Chr Marker Genotype S/S S/SHR SHR/SHR P Value (one-way ANOVA) SBP, mmhg 3 D3Rat D8Rat D9Rat Adjusted heart wt, mg 3 D3Rat53 1, , , * D8Rat24 1, , , D9Rat26 1, , , Body wt, g 4 D4Rat D12Rat D18Mgh D20Rat Values are means SE. S, S rat allele; SHR, SHR allele; ANOVA, analysis of variance. *Adjusted heart weight data on chr 8 did not meet the screening criteria for a QTL, but it is shown for the sake of comparison with blood pressure QTL data.

5 GENOME SCAN FOR BLOOD PRESSURE COMPARING S AND SHR 37 Fig. 2. Heart weight adjusted for differences in body weight is plotted against blood pressure (r 0.79, P ) for the F 2 (S SHR) population fed 8% NaCl for 4 wk. established chr 3 maps in the case of Clark et al. (5), these data on SHR crosses are difficult to interpret. A blood pressure QTL in the central part of chr 8 has been reported in crosses of normotensive strains with SHR (37, 38) or with S (15). In addition, a congenic strain substituting the QTL region was inadvertently produced when the gene for polydactyly-luxate syndrome (Lx), which is on chr 8, was moved from Brown Norway (BN) to the SHR background. The congenic strain had a blood pressure about 20 mmhg lower than SHR (21). Thus the blood pressure QTL on chr 8 observed here may be the same as seen in other studies. If so, this implies the existence of multiple functionally variant alleles at the QTL on chr 8, because S is the plus allele relative to SHR in the present work, and SHR is the plus allele relative to BN in the published work. Thus the order of the allelic effects on blood pressure must be S SHR BN. A blood pressure QTL on rat chr 9 was previously reported by us in an F 2 (S R) population (36). The position of that previously described QTL is shown in Fig. 1 as 1- and 2-LOD intervals along with 1- and 2-LOD intervals for the QTL from the present F 2 (S SHR) population. The 2-LOD intervals overlap, but the 1-LOD intervals do not (Fig. 1). It is impossible to be certain from these data whether the QTL from the different crosses are the same QTL or not. The issue can only be resolved by the construction of congenic strains and congenic substrains with progressively smaller chromosomal segments to localize the QTL to a small region (35). In comparing S and SHR strains directly on a high-salt diet, the higher blood pressure of S (Table 1) may be mainly due to the effect of chr 9, since the S allele has a large positive effect and the opposing effects of chr 3 and 8 will cancel each other out (Table 3). The corollary to knowing what QTL differentiate S from SHR is to know what QTL are likely to function similarly to increase blood pressure in the two strains. For example, a strong QTL signal on chr 10 in the region the angiotensin converting enzyme locus has been seen in many crosses involving SHR with a normotensive strain (17, 18, 20, 27, 28, 40) and also in crosses of S with a normotensive strain (10, 11, 15, 19). In the case of Dahl rats, the chr 10 QTL has been confirmed and further localized by the use of congenic strains (12, 15). Since there was no QTL observed on chr 10 in the present F 2 (S SHR) population, there must not be any allelic variation influencing blood pressure between S and SHR at the chr 10 QTL. Alternately, the failure to detect the chr 10 QTL here could be a false-negative, but this seems less likely as the chr 10 QTL has been easily detected in many studies quoted above. Similarly there are many articles describing the existence of blood pressure QTL in both S and SHR on chr 1, 2, and 13 [see Rapp (32) for a thorough review of this literature]. Thus it seems likely that the QTL on these chromosomes are also not functionally polymorphic between S and SHR, because no QTL signal was detected here for these chromosomes. In this article we have documented the expected correlation between heart weight (adjusted for body weight) and blood pressure. Because the heart hypertrophies when pumping chronically against the higher peripheral resistance present with increased blood pressure, heart weight serves as a check on the blood pressure measurements. Since blood pressure measurements by the tail cuff method are obtained under a condition of stress, it is reasonable to document their biological relevance. From our previous work we know that, for rats on high-salt diet, an early blood pressure measurement by the tail cuff method is a strong predictor of subsequent survival time (4). In the F 2 population there was a strong correlation of blood pressure and adjusted heart weight (Fig. 2). For QTL on chr 3 and 9, there were significant changes in heart weight associated with the blood pressure effect of the QTL. For the QTL on chr 8, the effect on heart weight was minimal and not significant. One speculation is that the QTL on chr 8 causes blood pressure changes only late in the experiment and these changes were not present sufficiently long to impact on heart weight. We thank Dr. Gary Churchill (Jackson Laboratories, Bar Harbor ME) for help with the statistical evaluation of genetic interactions. 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