Integrative genomic analysis of blood pressure and related phenotypes in rats.

Despite remarkable progress made in human genome-wide association studies, there remains a substantial gap between statistical evidence for genetic associations and functional comprehension of the underlying mechanisms governing these associations. As a means of bridging this gap, we performed genomic analysis of blood pressure (BP) and related phenotypes in spontaneously hypertensive rats (SHR) and their substrain, stroke-prone SHR (SHRSP), both of which are unique genetic models of severe hypertension and cardiovascular complications. By integrating whole-genome sequencing, transcriptome profiling, genome-wide linkage scans (maximum n=1415), fine congenic mapping (maximum n=8704), pharmacological intervention and comparative analysis with transcriptome-wide association study (TWAS) datasets, we searched causal genes and causal pathways for the tested traits. The overall results validated the polygenic architecture of elevated BP compared with a non-hypertensive control strain, Wistar Kyoto rats (WKY); e.g. inter-strain BP differences between SHRSP and WKY could be largely explained by an aggregate of BP changes in seven SHRSP-derived consomic strains. We identified 26 potential target genes, including rat homologs of human TWAS loci, for the tested traits. In this study, we re-discovered 18 genes that had previously been determined to contribute to hypertension or cardiovascular phenotypes. Notably, five of these genes belong to the kallikrein-kinin/renin-angiotensin systems (KKS/RAS), in which the most prominent differential expression between hypertensive and non-hypertensive alleles could be detected in rat Klk1 paralogs. In combination with a pharmacological intervention, we provide in vivo experimental evidence supporting the presence of key disease pathways, such as KKS/RAS, in a rat polygenic hypertension model.

Two genomic regions of chromosomes 1 and 18 explain most of the stroke susceptibility under salt loading in stroke-prone spontaneously hypertensive rat/Izm.

To clarify the genetic mechanisms of stroke susceptibility in the stroke-prone spontaneously hypertensive rat (SHRSP), a quantitative trait locus (QTL) analysis was performed. Using 295 F2 rats of a cross between SHRSP/Izm and SHR/Izm, 2 major QTLs for stroke latency under salt loading were identified on chromosomes (chr) 1 and 18. Evaluation of 6 reciprocal single and double congenic rats for these QTLs showed that substitution of the SHRSP for the SHR fragment at the chr 1 and 18 QTLs increased the relative risk for stroke by 8.4 and 5.0, respectively. The combined effect of the 2 QTLs was 10× greater than that of the background genome (by Cox hazard model). Blood pressure monitoring by radio telemetry indicated that the combination of the 2 QTLs had a clear effect on the salt-dependent blood pressure increase, suggesting an important role for the salt-sensitive blood pressure increase in the susceptibility of SHRSP to stroke. A haplotype analysis of 11 substrains of SHRSP and SHR using 340 simple sequence repeat markers in the chr 1 QTL suggested that the 7-Mbp fragment between D1Rat260 and D1Rat178 was most likely to harbor the responsible gene(s), which was confirmed by a study of additional subcongenic strains. This study indicated a major role for 2 QTLs on chr 1 and 18 in stroke susceptibility in SHRSP under salt loading. The salt-sensitive blood pressure increase was implied to play a key role in the stroke susceptibility.

Congenic removal of a QTL for blood pressure attenuates infarct size produced by middle cerebral artery occlusion in hypertensive rats.

A genome-wide screen found a blood pressure quantitative trait locus (QTL) on rat chromosome 1 in stroke-prone spontaneously hypertensive rats of a Japanese colony (SHRSP/Izm). In the present study, we investigated the effects of congenic removal of this QTL from SHRSP/Izm on infarct size produced by middle cerebral artery (MCA) occlusion. To establish the congenic strain (SHRSPwch1.0), the blood pressure QTL was introgressed from Wistar-Kyoto (WKY)/Izm to SHRSP/Izm by repeated backcrossing. Male SHRSP/Izm [10-12 wk old (young adult) n = 8, 5 mo old (adult) n = 17] and SHRSPwch1.0 (young adult n = 7, adult n = 15) were randomly assigned to distal MCA occlusion. Resting mean arterial blood pressure (MABP) was 212 +/- 23 mmHg in adult SHRSPwch1.0, which was significantly lower than 241 +/- 22 mmHg in SHRSP/Izm. Infarct volume in the congenic rats was significantly decreased compared with that in SHRSP/Izm (66.4 +/- 21.5 mm(3) vs. 103.4 +/- 24.8 mm(3)). Cerebral blood flow (CBF), determined at collaterally-perfused cortex with laser-Doppler flowmetry after MCA occlusion, was significantly greater in adult SHRSPwch1.0 compared with CBF in adult SHRSP/Izm. In young adult rats, there were no significant differences in MABP or in infarct volume between SHRSPwch1.0 and SHRSP/Izm. The congenic removal of a blood pressure QTL lowered blood pressure and caused a substantial reduction in infarct volume (-36%) with increased collateral CBF after MCA occlusion in the congenic rat. We demonstrated for the first time that the congenic strategy is useful to investigate the effects of genetic hypertension on focal ischemia or stroke.

Comprehensive QTL analysis of serum cholesterol levels before and after a high-cholesterol diet in SHRSP.

The stroke-prone spontaneously hypertensive rat (SHRSP) showed an exaggerated response to a high-fat, high-cholesterol (HFC) diet, and the resulting reactive hypercholesterolemia was suggested to exacerbate the atherogenic process in this rat. We thus performed a quantitative trait locus (QTL) analysis on the serum cholesterol level of SHRSP before and after the HFC diet, with the final goal being the identification of the genetic mechanisms of its reactive hypercholesterolemia. Three hundred fifty-eight F2 rats between SHRSP and Wistar-Kyoto rat were employed in the study. The serum cholesterol and apoprotein E were measured before and after 2 wk of feeding with the HFC diet. Multiple QTLs for the basal cholesterol level were identified on chromosomes 1 and 5, whereas those for the postdietary cholesterol level were on chromosomes 7, 15, and 16. The cholesterol QTLs before and after HFC diet did not overlap with one another, implying that the involved metabolic processes were considerably different between the two conditions. Supporting this, VLDL and LDL cholesterol were the major components of the postdietary serum cholesterol, whereas the basal cholesterol level consisted mainly of HDL cholesterol. A substantial difference of the QTLs between males and females was observed, especially after the HFC diet. The QTL on chromosome 15 had an inverse effect on the cholesterol level, suggesting that the congenic substitution of the SHRSP fragment with that of Wistar-Kyoto rats could induce a greater cholesterol level in SHRSP. This observation is significant in establishing a new model for atherosclerosis with hypertension in rats.

Exaggerated response to cold stress in a congenic strain for the quantitative trait locus for blood pressure.

OBJECTIVE: Stroke-prone spontaneously hypertensive rats (SHRSP) are known to have sympathetic hyperactivity to various stimuli. In the search for 'intermediate phenotypes' inferring the function of hypertension genes, the present study assessed responsiveness to cold stress in a congenic strain derived from SHRSP/Izm and Wistar-Kyoto/Izm (WKY/Izm). DESIGN: A congenic strain, WKYpch1.0, was established by 10 generations of backcrossing to transfer the chromosomal fragment between D1Wox29 and D1Arb21 of SHRSP to WKY. This fragment covered the 100:1 confidence interval of the quantitative trait locus (QTL) for blood pressure identified in a previous study. Response to cold stress was studied by exposing rats to 4 degrees C for 4 h. Blood pressure was monitored with telemetry. Urine was collected during the exposure, and urinary concentrations of catecholamines were measured by high-performance liquid chromatography. RESULTS: Under the cold stress, urinary excretion of norepinephrine (NE) and vanillylmandelic acid (VMA), as well as the plasma level of NE, was significantly greater in WKYpch1.0 than in WKY. The increase in blood pressure during the cold stress was also greater in WKYpch1.0 than in WKY. Further, neonatal chemical sympathectomy using guanethidine abolished the exaggerated response in blood pressure and in urinary excretion of NE and VMA in WKYpch1.0. CONCLUSION: These results suggested that the QTL region on rat chromosome 1 harbored genes responsible for the exaggerated response of the sympathetic nervous system to the cold stress. The relationship of this with the pathogenesis of hypertension should be elucidated in future studies.

Exaggerated response to restraint stress in rats congenic for the chromosome 1 blood pressure quantitative trait locus.

1. To understand the roles of a putative hypertension gene in the chromosome 1 quantitative trait locus (QTL) region, the response to restraint stress was studied in strains congenic for this QTL. 2. To establish congenic strains, the QTL region was introgressed from stroke-prone spontaneously hypertensive rats (SHRSP)/Izm to Wistar-Kyoto/Izm (WKY/Izm) rats by repeated backcrossing. Two congenic strains (WKYpch1.0 and WKYpch1.1) were established to cover the whole QTL region between D1Wox29 and D1Arb21 (approximately 40 cM) and a smaller region between D1Smu11 and D1Arb21 (approximately 10 cM), respectively. After telemetry probes were implanted, rats were exposed to restraint stress to investigate the blood pressure response. 3. Basal blood pressure measured by radiotelemetry differed significantly between WKY rats and WKYpch1.0 (103 +/- 10 and 116 +/- 4 mmHg, respectively; P = 0.002 by anova). When exposed to restraint stress, WKYpch1.0 showed a greater increase in blood pressre than did WKY rats. The exaggerated response in the WKYpch1.0 strain was abolished by chemical sympathectomy using guanethidine. The WKYpch1.1 rats did not differ significantly from WKY rats either in basal blood pressure or in the response to restraint stress. 4. In conclusion, a QTL for high blood pressure was successfully introgressed in the established congenic strain, WKYpch1.0. A gene (or genes) in the chromosome 1 QTL region modulates the cardiovascular responses to restraint stress in these congenic rats, probably through the sympathetic nervous system.

Genome-wide searches for blood pressure quantitative trait loci in the stroke-prone spontaneously hypertensive rat of a Japanese colony.

OBJECTIVES: Although several quantitative trait loci for blood pressure have been reported in stroke-prone spontaneously hypertensive rats (SHRSP), the results are not always concordant among different crosses. To evaluate potential confounding factors in linkage analysis, we performed genome-wide screens in F2 populations derived from SHRSP and Wistar-Kyoto rats of a Japanese colony. METHODS: Two F cohorts were independently produced: F2-1 (110 male and 110 female rats), and F2-2 (174 male and 184 female rats). Blood pressure was measured longitudinally (from 2 to 5 months of age and 1 month after salt-loading) in F2-1, while it was measured at 13 weeks of age in F2-2. Subsequent to an initial screen with 251 markers in F2-1 male progeny, 170 markers were selected and characterized in the remaining populations. RESULTS: When 578 rats were analyzed together, markers from five chromosomal regions showed significant linkage to blood pressure at 13 weeks of age. The strongest and the most consistent linkage was found on rat chromosome 1 (a maximal log of the odds score reached 8.3). In the other regions, the degree of linkage was more prominent in either of sexes. Some evidence of age-specific and sex-specific linkage was detected in five additional regions in the F2-1 cohort. In the Japanese colony, however, there was no significant linkage to several chromosomal regions previously reported in other SHRSP colonies. CONCLUSIONS: Our data provide solid evidence of a chromosome-1 linkage and demonstrate the importance of aging, sex, and dietary manipulation in linkage analysis. Also, the combination of parental rat strains seems to be critical when searching for blood pressure quantitative trait loci.

Fine linkage mapping of the blood pressure quantitative trait locus region on rat chromosome 1.

To narrow the area known to contain the blood pressure quantitative trait locus (QTL) on rat chromosome 1, we constructed a fine linkage map covering the blood pressure OTL region on the chromosome using 22 genetic markers informative for stroke-prone spontaneously hypertensive rats of the Izumo colony (SHRSP/Izm) and Wistar-Kyoto rats of the Izumo colony (WKY/Izm). Linkage mapping was done by genotyping 626 backcrossed rats from matings between SHRSP/Izm and WKY/Izm. Nineteen genetic markers informative for the two strains were selected from public databases. Two markers were newly isolated by screening a rat genomic library. One marker was mapped using a restriction endonuclease polymorphism. The region between DlWox29 and D1Smu11 was covered with 22 informative markers placed every 0.6 cM on average. In addition, 6 physiological candidates for a hypertension gene were mapped in this region either by linkage or by radiation hybrid (RH) mapping. This information should be essential for the construction and analysis of congenic strains for this QTL region.