Quantitative trait loci with opposing blood pressure effects demonstrating epistasis on Dahl rat chromosome 3
Ana Palijan,
Julie Dutil and
Alan Y. Deng
Research Centre-Centre Hospitalier de lUniversité de Montreal CHUM, Hôtel Dieu, Montreal, Quebec H2W 1T8, Canada
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ABSTRACT
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Our previous linkage studies indicated that there might be a blood pressure (BP) quantitative trait locus (QTL) on chromosome 3 (Chr 3) contrasting between the Dahl salt-sensitive (S) strain and the Lewis (LEW) strain. To prove and then to narrow down the segment containing this QTL, five congenic strains have been generated by replacing various segments of the S rats with the homologous segments of the LEW rats. They are designated as S.L1, S.L2, S.L3, S.L4, and S.L5, respectively. S.L2, S.L3, S.L4, and S.L5 are substrains of S.L1, i.e., they contain substitutions of smaller sections within the large fragment defined by S.L1. The construction of these congenic strains was facilitated by a genome-wide marker screening process. BPs of the rats were measured by telemetry. S.L2 and S.L3 shared a fragment of Chr 3 in common and both showed a BP-lowering effect, indicating the existence of "-BP" QTL alleles from LEW compared with S. In contrast, S.L4 involves a section with no overlap with either S.L2 or S.L3, and S.L4 showed a BP significantly higher than that of S rats, indicating the presence of "+BP" QTL alleles from LEW compared with S. Interestingly, the combined effect of the -BP QTL and +BP QTL alleles was "-" in S.L1, implying that the "-" QTL is epistatic to "+" QTL.
genetic hypertension; rat chromosome 3; Lewis rat; congenic strains
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INTRODUCTION
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LINKAGE AND CONGENIC ANALYSES have led to the mapping of numerous quantitative trait loci (QTLs) for blood pressure (BP) in the rat (14). Our focus is on the mapping of BP QTLs using the Dahl salt-sensitive rat (S) (4). Cicila and coworkers (1) detected a linkage of the endothelin 3 (Edn3) locus on chromosome (Chr 3) to BP. Later, by using a congenic strain, they confirmed the presence of a BP QTL near Edn3 (2). In our initial studies, another QTL was located in a segment of Chr 3 in an F2(S x LEW) population (8). The location of this QTL was on the opposite end of the same chromosome and seemed to be distinct from the QTL found by Cicila and coworkers (1, 2). The proximal location of this second QTL was also found in several other linkage studies in F2 populations using various rat hypertensive models (2, 3, 11, 12, 19).
There are a variety of congenic strains reported for physical mapping of BP QTLs in S rats (2, 48, 13, 16, 18). So far, the BP-raising alleles are always associated with the hypertensive S rats, whereas contrasting normotensive strains always carried BP-lowering alleles, despite the linkage evidence indicating that some BP-raising alleles could come from normotensive strains as well (8, 9). Another intriguing question is whether the combined BP effect of two contrasting QTLs would cancel each other out, or whether one QTL could be dominant over the other.
The purpose of the present work is to address the following questions: 1) Is there a single QTL or multiple QTLs in the region in question on Chr 3?; and 2) If there are multiple QTLs, what are their BP effects, individually and in combination?
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METHODS
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Animals.
The SS/Jr rat used for making congenic substrains will be designated as S in the present report and is the same as we used previously (5, 7, 13, 18). To ensure that our S rats were not genetically contaminated, a rigorous and strict quality control procedure has been instituted. The quality control measures consisted of a genetic testing and a physical discrimination. For a detailed quality control procedure, please consult Ref. 7. LEW/CrlBR (LEW) rats were purchased from Charles River Laboratories (La Salle, Quebec, Canada). The same quality control as for the S strain applied to LEW also on the basis of genetic monitoring with genome-wide markers (7, Table 1). Protocols for handling as well as maintaining animals were approved by our institutional animal committee. All experimental procedures were in accordance with the guidelines of institutional, provincial, and federal regulations.
Breeding scheme for generating congenic strains.
The breeding procedure and screening protocol were essentially the same as reported previously (5, 13, 18). Briefly, rats of the S and LEW strains were bred to produce F1 rats, which were then backcrossed to S rats to produce the first backcross generation (BC1). BC1 rats were genotyped for 99 markers spaced throughout the rat genome with an average spacing of about 18 cM (Table 1, which is similar to that given in Refs. 5, 13, and 18). One BC1 rat was selected and designated as the ideal breeder, which was heterozygous SL for a Chr 3 region but possessed the maximum SS homozygosity for the rest of the untargeted genome. The ideal breeder BC1 was mated with an S rat to produce BC2. BC2 rats were screened exactly as for those of BC1 to derive an ideal breeder BC2. This process continued until BC5. At this point, it was found that only the markers delineating a Chr 3 region of interest (Fig. 1) were heterozygous SL; whereas the markers for the rest of the genome including those flanking the Chr 3 region of interest were homozygous SS.

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Fig. 1. Mapping of two opposing blood pressure (BP) quantitative trait loci (QTL) on Dahl rat chromosome 3 (Chr 3). The linkage map is essentially the same as published previously (8), which is based on an F2(S x LEW) population. Numbers to the right of the map are units in centimorgans (cM), and to the left of the map are units in centirays (cR) between markers. Solid bars under congenic strains symbolize the S chromosome fragments that have been replaced by that of the LEW rat. The entire region indicated by solid bars and junctions between the solid and open bars are homozygous LL on the map for all the markers listed in the corresponding positions. Open bars on ends of solid bars indicate the ambiguities of crossover breakpoints between markers. Junctions between solid and open bars as well as ends of chromosome regions of interest in each strain are connected by dotted lines to the marker positions on the map. The "physical map" refers to the alignment of rat supercontigs in an ascending order from top to bottom on Chr 3 obtained by blasting a marker to the rat genome database (http://www.ncbi.nlm.nih.gov/genome/seq/RnBlast.html). The name of a supercontig starts with NW_043 followed by three numbers. Italic digits to the left side of a contig in parentheses indicate its approximate size in base pairs. Markers are anonymous (http://www-genome.wi.mit.edu/rat/public/). Congenic strains are S.LEW-D3Rat52/D3Rat17 (S.L1), S.LEW-D3Rat52/D3Chm63 (S.L2), S.LEW-D3Rat52/D3Rat130 (S.L3), S.LEW-D3Chm64/D3Rat17 (S.L4), and S.LEW-D3Mco21/D3Rat17 (S.L5), respectively. Mean arterial pressures (MAPs) averaged throughout the period of measurements for the strains are as follows (in mmHg): S (175 ± 3), S.L1 (141 ± 4), S.L2 (146 ± 6), S.L3 (141 ± 4), S.L4 (195 ± 9), and S.L5 (166 ± 9). Average diastolic arterial pressures (DAPs) are as follows (in mmHg): S (150 ± 4), S.L1 (121 ± 5), S.L2 (128 ± 5), S.L3 (121 ± 4), S.L4 (167 ± 9), and S.L5 (140 ± 8). Average systolic arterial pressures (SAPs) are as follows (in mmHg): S (203 ± 4), S.L1 (164 ± 4), S.L2 (173 ± 8), S.L3 (167 ± 4), S.L4 (228 ± 8), and S.L5 (194 ± 9).
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To establish a congenic strain, a BC5 rat was mated to an S rat to duplicate the segment of interest. Subsequently, a female and a male rat were sister-brother bred to generate rats homozygous LL for the region of interest, but homozygous SS for the rest of Chr 3 and rest of the genome. In the process, five congenic strains were produced and are designated as S.LEW-D3Rat52/D3Rat17 (abbreviated as S.L1), S.LEW-D3Rat52/D3Chm63 (S.L2), S.LEW-D3Rat52/D3Rat130 (S.L3), S.LEW-D3Chm64/D3Rat17 (S.L4) and S.LEW-D3Mco21/D3Rat17 (S.L5). The chromosome regions homozygous LL in the strains are shown as solid bars in Fig. 1. All the markers in the region concerned were genotyped in the congenic strains.
BP measurements.
The determination of BPs of the rats is essentially the same as described previously (5, 7, 13, 18). In brief, the mating pairs of the S and congenic strains to be studied were bred simultaneously. Male rats were selected and weaned at 21 days of age, maintained on a low-salt diet (0.2% NaCl, Harlan Teklad 7034) and then fed a high-salt diet (2% NaCl, Harlan Teklad 94217) starting from 35 days of age until the end of the experiment. Telemetry probes were implanted when rats were 56 days old (i.e., after 3 wk of the high-salt diet) with their body weights between 250 and 320 g. After the surgery, the rats were allowed 10 days to recuperate before their BPs were read. The implantation of telemetry probes, the age, and postoperative cares of animal are the same as described before (5, 7, 13, 18). The telemetry system from Data Sciences (St. Paul, MN) was used to measure BP. Each telemetry probe was calibrated before and cleaned after each usage according to manufacturers instructions.
Production of new markers.
Supercontigs located in Chr 3 regions of interest were first identified by blasting with a known marker at the website, http://www.ncbi.nlm.nih.gov/genome/seq/RnBlast.html (Fig. 1). From such a supercontig, regions containing microsatellites were searched and, when found, were used to design markers for genotyping rats based on PCR. These new markers were designated with D3Chm prefixes, which represent the Centre Hospitalier de lUniversité de Montreal (CHUM). Only those that give workable PCR products are listed in Table 2.
DNA extraction and genotyping.
DNA for each rat was extracted by tail biopsy using a Qiagen genome kit, and the genotype of each rat was determined by PCR based on the methods previously published (5, 7, 13, 18).
Radiation hybrid mapping.
A rat/hamster (RH) panel of 96 radiation hybrids was purchased from Research Genetics (Huntsville, AL, http://www.resgen.com/). For chromosome mapping, each marker was genotyped using RH by PCR according to a previously published protocol (5, 7, 13, 18). To locate a marker of interest onto an existing RH framework map, the results of genotyping were entered into a web site (http://rgd.mcw.edu/RHMAPSERVER).
Statistical analysis.
Repeated measures analysis of variance (ANOVA) followed by the Dunnett test in the SYSTAT 9 program (SPSS, Chicago, IL) was used to compare the significance level for a difference or a lack of it in a BP component between a congenic strain and the S strain. The Dunnett test takes into account multiple group comparisons as well as sample sizes among the comparing groups. In the analysis, a BP component was compared at each day for the period of measurement among the strains (5, 7, 13, 18).
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RESULTS
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Congenic constructions.
By following the strategy of selecting an ideal BC rat at each generation, the construction of five congenic strains were accomplished in five, instead of eight, BC generations as used conventionally (15). The current results are consistent with that of other investigators (10) and with our previous work (5, 13, 18). All the genome-wide markers except the region of interest (Fig. 1) turned out to be homozygous at BC5. The chromosome regions containing various LEW substitutions in the congenic rats are shown in Fig. 1. In the end, each region of interest from the S rat was replaced by the homologous region of the LEW rat, i.e., a LL region on the SS genetic background.
Marker generations.
Most of the markers obtained from the rat database at http://www-genome.wi.mit.edu/rat/public/no_frames.html were screened for polymorphisms between S and LEW, which span the region between D3Rat52 and D3Mgh11, and the region between D3Rat52 and the telomere at the top. Large gaps remained on the map. To more precisely define crossover regions in the congenic strains, more polymorphic markers are needed. By taking advantage of the recently released rat sequence (http://www.ncbi.nlm.nih.gov/genome/seq/RnBlast.html), we produced markers originating from the supercontigs located in the Chr 3 region of interest (see METHODS for details). These markers are listed in Table 2. Some of these markers are polymorphic and have been used to define chromosome crossovers in congenic strains (Fig. 1).
BP studies.
The basic design of raising animals is similar to our previous congenic work regarding the age and sex and in terms of the timetable of dietary treatments (5, 7, 13, 18). All the BP components were measured including mean arterial (MAP), diastolic (DAP), and systolic pressures (SAP). For the simplicity of presentation, each point in the graphs in Fig. 2 represents averaged 24-h readings taken from averaged values at every 4 h. One BP reading was taken every 4 h for all the data points collected within that period of time. Then, these 4-h readings were averaged for 24 h or six readings were averaged within 24 h to obtain one data point, which appears as a point on the graph. Please see Dutil and Deng (7) for detailed comparisons in BPs of S and a congenic strain showing hour-to-hour readings including diurnal variations.

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Fig. 2. Comparisons in MAP (bottom), DAP (middle), and SAP (top) pressures between the S.L1, S.L2, S.L3, S.L4, and S.L5 congenic strains and the S strain. Each time point on the graph represents an average of 24-h readings. S, the Dahl salt-sensitive strain. Each point on the 24-h graph is an average of readings taken at every 4 h. Error bars represent SE; n = number of rats. For strain designations, see the legend for Fig. 1.
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Mapping of BP QTLs.
Figure 2 shows comparisons of MAP, DAP, and SAP of S rats with those of congenic rats. Congenic strains were compared with each other in the length of the chromosome replacements and for their effects on BPs. The region containing the QTL can be localized to the segment shared in two strains that showed significant changes in BPs. MAP, DAP, and SAP values of S.L2 and S.L3 were lower (P < 0.04) than those of the S strain (Fig. 2, DF). In contrast, MAP, DAP, and SAP values of S.L4 were higher (P < 0.03) than those of the S strain (Fig. 2, DF). MAP, DAP, and SAP values of S.L1 were lower (P < 0.04) than those of the S strain (Fig. 2, AC) but were not different from those of S.L2 and S.L3 (P > 0.5). MAP, DAP, and SAP values of S.L5 were not significantly different (P < 0.10) from those of the S strain (Fig. 2, AC).
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DISCUSSION
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The major findings of the current work are as follows: 1) two BP QTLs are located adjacent to each other, one exerting a BP-lowering effect, i.e., a "-BP" QTL, and the other exerting a BP-raising effect, i.e., a "+BP" QTL; and 2) the -BP QTL is epistatic to the +BP QTL.
A BP QTL is mapped to the Chr 3 segment between D3Rat52 and D3Rat130 (Fig. 1), which is shared between S.L2 and S.L3 congenic strains. LEW rats carry BP-decreasing alleles of this QTL compared with those of S rats. For the sake of presentation, this QTL is designated as a -BP QTL. Another QTL is found between D3Chm63 and D3Ra26 because S.L4 has a BP effect whereas S.L5 does not (Fig. 2, AC). In contrast to possessing a -BP QTL, LEW rats, being a normotensive strain, also carry BP-increasing alleles from this second QTL. For the sake of presentation, this second QTL is designated as a +BP QTL. The presence of a +BP QTL in LEW indicates that being a normotensive strain does not mean it exclusively carries BP-lowering alleles.
Then it is logical to pose the question "What is the relationship between a -BP and a +BP QTLs?" Contrary to a conventional prediction that one -BP and one +BP QTL would cancel out the effect of each other, the combined effect of the -BP and the +BP QTLs results actually in an overall decrease in BP as shown in S.L1 (Fig. 1 and Fig. 2, AC). Interestingly, the BP-lowering consequence in S.L1 is not different (P > 0.50) from that either in S.L2 or S.L3. This fact further supports the notion that relationships among the QTLs in question are epistatic, although more complex interactions among QTLs from other chromosomes cannot be ruled out.
The possibility cannot be excluded that more than one BP QTL is located in each of the segments defined by S.L3 and S.L4 congenic strains. The current work has practical implications in dissecting BP QTLs as well. When a subsegment in a large chromosome region is found to possess the same BP effect as the large region, it is also possible that other subsegments untested within the large chromosome region could harbor additional BP QTL(s). The only way to rule out this possibility is to construct a congenic strain to include that untested region, which consequently does not show any BP effect.
The region containing the -BP QTL in the rat shares a conserved synteny relationship with a segment of mouse Chr 2, and segments of human Chrs 9 and 2. The region harboring the +BP QTL is homologous to a fragment of mouse Chr 2 and to sections of human Chrs 2, 11, and 20 (http://www.well.ox.ac.uk/rat_mapping_resources/Comparative_maps/compa_map_chr03.html). A fine mapping of the QTLs in question may further refine the exact portion of the human chromosome with which it has a conserved synteny and eventually help unravel some of the genetic determinants in human hypertension. As the genome projects for the human, the mouse, and the rat progress, the genomic information will converge and might also contribute to the identification of the BP QTL in the rat.
These two QTLs are located at the opposite end of Chr 3 (Fig. 1), where Cicila and coworkers (1, 2) found another QTL. Other investigators found a possible QTL for cardiac mass independently of BP (17), and their QTL region is near that for the +BP QTL in our current work (Fig. 1). In the present and our other works, BP effects of QTLs are often associated with those of the cardiac mass, also (4, 5, 7, 13, 18). It remains to be determined whether certain BP QTLs in S rats could be separated from a QTL for the cardiac mass.
In summary, a case of epistasis between two QTLs in close proximity is strongly supported by the use of congenic strains. Up to date, such an epistatic interaction was limited to two QTLs with BP effects in the same direction, i.e., both lowering BP by the QTL alleles from a normotensive strain (6, 16). Our current work adds further complexity to epistatic interactions to include two QTLs on Dahl Chr 3 with opposing BP effects by QTL alleles from a normotensive strain, i.e., one lowering whereas the other raising BP. This phenomenon supports the hypothesis that to achieve a threshold in limiting BP in a normotensive strain, it can be accomplished not only by having a higher proportion of BP-lowering QTL alleles, but also by epistatic interactions between BP QTLs in a sense of one having dominance over the other.
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DISCLOSURES
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This work was supported by grants from American Heart Associate National Center, the Kidney Foundation of Canada, and Canadian Institutes for Health Research (CIHR) to A. Y. Deng. A. Y. Deng is an Established Investigator of the American Heart Association. J. Dutil is a holder of a CIHR/Canadian Society of Hypertension studentship.
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ACKNOWLEDGMENTS
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We thank Dr. Pavel Hamet for a critical reading of the manuscript.
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FOOTNOTES
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Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).
Address for reprint requests and other correspondence: A. Y. Deng, Research Centre, Centre Hospitalier de lUniversité de Montreal (CHUM), 7-132 Pavillon Jeanne Mance, 3840, rue St. Urbain, Montreal, Quebec H2W 1T8, Canada (E-mail: alan.deng{at}umontreal.ca).
10.1152/physiolgenomics.00084.2003.
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