Two linked blood pressure quantitative trait loci on chromosome 10 defined by dahl rat congenic strains.

Aquantitative trait locus (QTL) for blood pressure was previously detected on rat chromosome 10 (RNO10) by linkage analysis and confirmed by the construction of congenic strains that encompass large regions of RNO10. In the present study, the rat RNO10 blood pressure QTL was dissected by the further construction of congenic substrains. The original congenic region was shown to contain 2 blood pressure QTLs (QTL 1 and QTL 2) approximately 24 cM apart. These were localized to a <2.6-cM region between markers D10Rat27 and D10Rat24 for QTL 1 and to a <3.2-cM region between D10Rat12 and D10Mco70 for QTL 2. Comparative mapping suggests that the rat RNO10 QTL 2 could be localized very close to a blood pressure QTL described by sib-pair analysis on human chromosome 17, but this is not definitively established because of multiple and complex chromosomal rearrangements between rodents and humans.

High-resolution mapping of the blood pressure QTL on chromosome 7 using Dahl rat congenic strains.

It was previously shown using Dahl salt-sensitive (S) and salt-resistant (R) rats that a blood pressure quantitative trait locus (QTL) was present on rat chromosome 7. In the present work, this QTL was localized to a region less than 0.54 cM in size on the linkage map using a series of congenic strains. This region was contained in a single yeast artificial chromosome that was 220 kb long. This small segment still contained the primary candidate locus Cyp11b1 (11beta-hydroxylase), but the adjacent candidate genes Cyp11b2 (aldosterone synthase) and Cyp11b3 were ruled out. It is concluded that 11beta-hydroxylase, through its known genetic variants altering the production of 18-hydroxy-11-deoxy corticosterone, is very likely to account for the blood pressure QTL on chromosome 7 in the Dahl rat model of hypertension. This QTL accounts for about 23 mm Hg under the condition of 2% NaCl diet for 24 days.

Multiple blood pressure QTL on rat chromosome 1 defined by Dahl rat congenic strains.

A series of congenic strains were constructed in which segments of chromosome (chr) 1 from Lewis (LEW) rats were introgressed into the Dahl salt-sensitive (S) strain. Three blood pressure quantitative trait loci (QTL) were defined. Two of these (QTL 1a and QTL 1b) were closely linked in the region between 1q31 and 1q35. The third blood pressure QTL (QTL region 2) was close to the centromere between 1p11 and 1q12, which includes the candidate gene Slc9a3 for sodium/hydrogen exchange. The blood pressure QTL 1a and QTL 1b defined here overlap significantly with QTL for disease phenotypes of renal failure, stroke, ventricular mass, and salt susceptibility defined in other rat strains, implying that these disease phenotypes and our blood pressure phenotype have causes in common. QTL 1b also corresponded approximately with a blood pressure QTL described on human chr 15. The QTL region 2 corresponded approximately with blood pressure QTL described on mouse chr 10 and human chr 6.

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

Our purpose was to define quantitative trait loci (QTL) for blood pressure that differ between two widely used hypertensive rat strains, the Dahl salt-sensitive (S) rat and the spontaneously hypertensive rat (SHR). A genome scan was done on an F(2) (S x 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.

Genetic analysis of inherited hypertension in the rat.

Blood pressure is a quantitative trait that has a strong genetic component in humans and rats. Several selectively bred strains of rats with divergent blood pressures serve as an animal model for genetic dissection of the causes of inherited hypertension. The goal is to identify the genetic loci controlling blood pressure, i.e., the so-called quantitative trait loci (QTL). The theoretical basis for such genetic dissection and recent progress in understanding genetic hypertension are reviewed. The usual paradigm is to produce segregating populations derived from a hypertensive and normotensive strain and to seek linkage of blood pressure to genetic markers using recently developed statistical techniques for QTL analysis. This has yielded candidate QTL regions on almost every rat chromosome, and also some interactions between QTL have been defined. These statistically defined QTL regions are much too large to practice positional cloning to identify the genes involved. Most investigators are, therefore, fine mapping the QTL using congenic strains to substitute small segments of chromosome from one strain into another. Although impressive progress has been made, this process is slow due to the extensive breeding that is required. At this point, no blood pressure QTL have met stringent criteria for identification, but this should be an attainable goal given the recently developed genomic resources for the rat. Similar experiments are ongoing to look for genes that influence cardiac hypertrophy, stroke, and renal failure and that are independent of the genes for hypertension.

Body fluid expansion is not essential for salt-induced hypertension in SS/Jr rats.

To evaluate the importance of volume in the development of hypertension in inbred Dahl salt-sensitive rats (SS/Jr), we measured the changes in blood pressure (BP) that occurred with oral intake of food (salt) and water in rats whose body weight was permitted to increase versus those in which body weight was maintained constant with a servo-control system. We hypothesized that if volume expansion is essential in the development of hypertension, then BP would not increase if body weight was held constant. We found that oral presentation of chow containing 4% salt to SS/Jr rats caused BP to increase 32.2 +/- 2.9 mmHg over 4 days when body weight was controlled at its initial value. Plasma sodium increased from 142.0 to 145.2 meq/l during 4 days of high salt. Neither plasma volume, hematocrit, nor central venous pressure changed significantly on the high-salt diet. In contrast, the inbred Dahl salt-resistant rats (SR/Jr) did not increase their BP during body weight control when given 4% salt. This demonstrates that volume expansion is not an obligatory step in the pressure response to increased salt in SS/Jr rats. Our results obtained with oral presentation of salt, in contrast to intravenous, represent a physiological evaluation of the significance of volume changes in response to dietary salt because no potential regulatory reflexes have been bypassed.

A gene map of the rat derived from linkage analysis and related regions in the mouse and human genomes.

We report the localization by linkage analysis in the rat genome of 148 new markers derived from 128 distinct known gene sequences, ESTs, and anonymous sequences selected in GenBank database on the basis of the presence of a repeated element. The composite linkage map of the rat contributed by our group integrates mapping information on a total of 370 different known genes, ESTs, and anonymous mouse or human sequences, and provides a valuable tool for comparative genome analysis. 206 and 254 homologous loci were identified in the mouse and human genomes respectively. Our linkage map, which combines both anonymous markers and gene markers, should facilitate the advancement of genetic studies for a wide variety of rat models characterized for complete phenotypes. The comparative genome mapping should define genetic regions in human likely to be homologous to susceptibility loci identified in rat and provide useful information for the identification of new potential candidates for genetic disorders.

Strain differences in neointimal hyperplasia in the rat.

We performed an initial screen of 11 rat strains by use of a standard balloon injury to the left iliac artery to observe whether genetically determined differences existed in the development of neointimal hyperplasia. Neointimal hyperplasia was assayed 8 weeks after the vascular injury on coded microscopic sections. Statistically significant differences in the percentages of the vascular wall cross-sectional areas composed of intima (percentage intima) secondary to neointimal hyperplasia were noted among the different rat strains (P<0.02), with the Brown-Norway (BN), Dark Agouti, and Milan normotensive strain rats having the highest and the spontaneously hypertensive rats (SHR) having the lowest percentages of intima. In a separate experiment, F1 hybrids of SHRxBN strains and parental BN and SHR underwent the vascular injury, and the parental strains again showed a statistically significant difference from one another in the mean percentage of intima (P<0. 0001). The F1 hybrids showed an average percentage of intima intermediate between those of the parental strains. The average lumen size of the injured BN vessels were significantly smaller than that of the noninjured control vessels (P=0.044), but this significance disappeared when the circular areas of these vessels were calculated without taking neointimal growth into consideration (P=0.649). These results provide the groundwork for a genetic linkage analysis to identify the genes that influence the development of neointimal hyperplasia after vascular injury.

Complete genome searches for quantitative trait loci controlling blood pressure and related traits in four segregating populations derived from Dahl hypertensive rats.

The Dahl salt-sensitive rat is one of the principal animal models of hereditary hypertension. Genome-wide searches were undertaken to detect quantitative trait loci (QTLs) that influence blood pressure, cardiac mass, and body weight in four F2 populations derived from Dahl salt-sensitive rats and different inbred normotensive control strains of rat. We detected three QTLs associated with one or more of the phenotypes, using a stringent statistical criterion for linkage (p < 0.00003). These included a novel QTL linked to blood pressure on rat Chromosome (Chr) 12, and another QTL on rat Chr 3 linked to body weight. A QTL on rat Chr 10 for which linkage to blood pressure has been described in other crosses was found to be a principal determinant of blood pressure and cardiac mass in some but not all of the crosses examined here. Three other regions showed evidence of linkage to these phenotypes with a less stringent statistical criterion of linkage at QTLs previously reported in other studies. As part of our study, microsatellite markers have been developed for three candidate genes for investigation in hypertension, and the genes have been localized by linkage mapping. These are: the rat Gs alpha subunit (Gnas) gene, the alpha-1B adrenergic receptor (Adra1b), and the Na+, K+-ATPase beta2 subunit (Atp1b2) gene.

Two blood pressure/cardiac mass quantitative trait loci on chromosome 3 in Dahl rats.

Interval mapping was used to identify putative quantitative trait loci (QTL) for blood pressure and cardiac mass on Chromosome (Chr) 3 in F1(S x R) x S population of 150 rats raised on an 8% NaCl diet. Two genetic markers 95.7 cM apart, D3Wox3 and D3Mco5 (tightly linked to Edn3), showed "suggestive" linkage to blood pressure (LOD = 2.0 and 1.8 respectively). In addition, D3Wox3 showed "suggestive" linkage to heart weight (LOD = 2.5), and D3Mco5 showed "suggestive" linkage to body weight-adjusted heart weight (LOD = 2.1). Congenic rats (designated S.R-Edn3) were constructed by introgressing the R-rat Edn3 allele (and flanking loci) into the S strain. On a 2% NaCl diet, S.R-Edn3 rats had lower blood pressure (21.4 mm Hg, P = 0. 0005) and heart weight (59 mg, P = 0.0038) compared with S rats, confirming the existence of a blood pressure QTL on Chr 3 near Edn3 even though QTL linkage analysis of blood pressure did not achieve stringent statistical criteria for significance. The results of the congenic experiment and the large distance between the two putative QTL suggest the presence of at least two independent blood pressure/cardiac mass QTL detectable on Chr 3 in the Dahl rat model of genetic hypertension.

Improved linkage map and thirty new microsatellite markers for rat Chromosome 10.

An improved linkage map for rat Chromosome (Chr) 10 with two F2 populations was constructed. Thirty new microsatellite markers were generated from a Chr 10-specific, small-insert genomic library and mapped to rat Chr 10. Among them were the rat homologs for the mouse gene for light and heavy chains of myeloperoxidase and human neurofibromatosis 1. Eight newly generated markers (D10Mco62, D10Mco63, D10Mco64, D10Mco65, D10Mco67, D10Mco68, D10Mco70, and D10Mco74) were mapped to the region of the rat Chr 10 blood pressure QTL. The availability of such markers may be instrumental in the search for genes responsible for the hypertension.

Localization of a blood pressure QTL on rat chromosome 1 using Dahl rat congenic strains.

We previously reported that markers on rat chromosome 1 are genetically linked to blood pressure in an F(2) population derived from Dahl salt hypertension-sensitive (S) and Lewis (LEW) rats. Because there was evidence for more than one blood pressure quantitative trait locus (QTL) on chromosome 1, an initial congenic strain introgressing a large 118-centimorgan (cM) segment of LEW chromosome 1 into the S background had been constructed. This initial congenic strain had a reduced blood pressure compared with S rats, proving the existence of a blood pressure QTL, but not giving a good localization of the QTL. In the present work a series of five overlapping congenic substrains were produced from the original congenic strain in order to localize a blood pressure QTL to a 25-cM region near the center of chromosome 1. The congenic substrains also ruled out the Sa locus as a blood pressure QTL in the S vs. LEW comparison because the Sa locus was contained in a congenic substrain that did not alter blood pressure.

Linkage mapping of fifty-eight new rat microsatellite markers.

Fifty-eight new anonymous simple sequence repeats (SSR) were generated and mapped to various rat chromosomes. Among them two genes (rat homologs for human cadherin-14 and mouse fibroblast growth factor-related protein) were mapped on Chromosomes (Chrs) 2 and 11 respectively. The majority of markers were generated from a small insert genomic library specific to Chr 11, 13, 14, and 15. Twenty new markers were mapped to Chr 13, which is known to contain a blood pressure quantitative trait locus (QTL). Several approaches to obtain microsatellite markers are described. The protocols and newly generated markers should be useful for ongoing rat genome project.

Linkage analysis and construction of a congenic strain for a blood pressure QTL on rat chromosome 9.

A blood pressure quantitative trait locus was found (LOD = 5.0) on rat chromosome 9 using a large F2 population (N = 233) derived from Dahl salt-sensitive (S) and Dahl salt-resistant (R) rats. The F2 rats were fed 8% NaCl diet for 8 weeks. A congenic strain introgressing the R low-blood-pressure QTL allele on chromosome 9 into the S strain was constructed. The congenic strain, designated S.R(chr 9), had a lower blood pressure (19 mm Hg, P < 0.0001) and lower heart weight (112 mg, P < 0.0001) than S rats (2% NaCl diet for 24 days), proving the existence of a blood pressure QTL in the congenic region of about 21 cM.

Genome scan and congenic strains for blood pressure QTL using Dahl salt-sensitive rats.

An F2 population (n = 151) derived from Dahl salt-sensitive (S) and Lewis rats was raised on a 8% NaCl diet for 9 weeks and analyzed for blood pressure quantitative trait loci (QTL) by use of a whole genome scan. Chromosomes 5 and 10 yielded lod scores for linkage to blood pressure that were significant; chromosomes 1, 2, 3, 8, 16, 17, and 18 gave lod scores suggestive for linkage. Chromosome 7 gave a significant signal for heart weight with a lesser effect on blood pressure. Congenic strains were constructed by introgressing Lewis low-blood-pressure QTL alleles for chromosomes 1, 5, 10, and 17 into the S genetic background. Congenic strains for chromosomes 1, 5, and 10 had significantly lower blood pressure than S, proving the existence of QTL on these chromosomes, but the chromosome 17 congenic strain failed to trap a contrasting QTL allele. The QTL allele increasing blood pressure originated from S rats for all QTL except those on chromosomes 2 and 7 in which the Lewis allele increased blood pressure. Interactions between each QTL and every other locus in the genome scan yielded significant interactions between chromosomes 10 and 4, and between chromosomes 2 and 3.

Practicable approaches to targeted comparative mapping of rat chromosome regions: linkage mapping of five genes on rat chromosome 13.

We demonstrate feasible approaches to comparative mapping between the region near the renin locus on rat chromosome 13 and human chromosome 1q by assigning five additional genes as anchor loci. Base-substitution polymorphisms were sought in the 3'- and/or 5'-untranslated regions for subsequent development of PCR-amplified markers, which, in turn, could be used for either restriction fragment analysis or allele-specific PCR.

An improved linkage map of rat chromosome 3 with three mapping panels.

Our purposes were to develop an improved linkage map for rat Chromosome 3 and to develop new markers polymorphic between Dahl salt-sensitive (S) and Dahl salt-resistant (R) rats. The linkage mapping panel consisted of three F2 populations totaling 359 rats. Twenty-five new markers were developed and placed on the linkage map. About half of these markers (13) were polymorphic between S and R rats. The final map spans 124.7 centiMorgans (cM) and includes 64 markers. The average distance between adjacent markers is 1.9 cM, and the largest separation is 10.5 cM.

Construction of a double congenic strain to prove an epistatic interaction on blood pressure between rat chromosomes 2 and 10.

Previously we presented suggestive evidence from an F2 segregating population for an interaction on blood pressure (BP) between quantitative trait loci (QTL) on rat chromosomes (Chr) 2 and 10. To prove the existence of such an interaction, we developed congenic strains for Chr 2 and 10 by introgressing the low BP QTL alleles into the Dahl salt-sensitive (S) strain. A double congenic strain was also constructed with both the Chr 2 and 10 low BP QTL alleles on the S background. The four strains (S, Chr 2 congenic, Chr 10 congenic, and Chr 2/10 double congenic) were studied for BP response to increased salt intake. An analysis of variance showed significant main effects of Chr 2, Chr 10, and a significant interaction between Chr 2 and 10 on BP and heart weight (all P < 0.0001). The interaction accounted for 24 mmHg of BP and 79 mg of heart weight. Thus, the discovery and proof of epistatic interactions are clearly critical to understanding the genetics of blood pressure.