Quantitative Trait Loci in Genetically Hypertensive Rats
Possible Sex Specificity
We performed a total genome screen in an F2 cross derived from the stroke-prone spontaneously hypertensive rat and the normotensive Wistar-Kyoto rat. Blood pressure at baseline and after 1% NaCl was measured by radiotelemetry; other phenotypes included heart rate, motor activity, left ventricle weight to body weight ratio, and vascular smooth muscle cell polyploidy, a measure of vascular hypertrophy. Quantitative trait loci affecting a given phenotype were mapped relative to microsatellite markers by using the mapmaker/qtl 1.1 computer package. We identified three blood pressure quantitative trait loci, two on rat chromosome 2 and one on rat chromosome 3. The quantitative trait loci close to genetic markers D2Mgh12 (“suggestive” linkage, with a maximal logarithm of the odds [LOD] score of 3.1) and D3Mgh16 (significant linkage, with a maximal LOD score of 5.6) showed possible sex specificity in the male F2 cohort only. This was confirmed by the likelihood ratio test for the difference in locus effects between the sexes. We also identified a new quantitative trait locus for LV hypertrophy on rat chromosome 14 (“suggestive” linkage, with a maximal LOD score of 3.1). The sex specificity of blood pressure quantitative trait loci will be important in designing congenic strains and substrains for fine genetic mapping and for identifying genes that regulate blood pressure.
The SHRSP is one of the best existing animal models of human essential hypertension and is characterized by a number of vascular complications (eg, end-organ damage) not dissimilar to those in human disease.1 2 Previous studies have confirmed that high BP in the SHRSP shows polygenic inheritance, and three QTLs have been identified in the first SHRSPHeidelberg×WKY cross: BP/SP-1 (or Bp1), a locus that has been mapped to rat chromosome 10; Bp2, a locus on chromosome 18; and BP/SP-2, a locus on the X chromosome.3 4 Further analysis and comparison of rat and human genetic maps have suggested the angiotensin converting enzyme gene as a possible candidate for BP/SP-1 (Bp1). Multilocus linkage analysis performed in one of two studies3 showed that the rat angiotensin converting enzyme gene fell within the 10:1 odds region from BP/SP-1 in the analysis of BP after salt loading (1% NaCl in the rats' drinking water) and within the 100:1 odds region for baseline BP phenotypes. More recent data on the SHRSPHeidelberg ×WKY congenic substrains revealed that BP/SP-1 can be dissected into two loci: BP/SP-1a, linked to basal BP, and BP/SP-1b, which cosegregates with BP after salt loading.5
These initial studies confirmed that the strategy that is best described as a total genome screen is directly applicable to detect QTLs in genetic models of hypertension.6 Recent published studies have provided a large number of newly developed microsatellite markers, the majority of which have been mapped to individual chromosome regions.7 8 9 These efforts to improve the density of the rat genetic linkage map, together with high-fidelity phenotypic measurements such as radiotelemetry direct hemodynamic monitoring, have allowed a new generation of mapping studies in hypertension.
The current study was designed to perform a total genome screen in the SHRSPGlasgow×WKY cross with multiple phenotypes, including direct systolic and diastolic BP at baseline and after salt loading, HR, and motor activity as measured by radiotelemetry as well as LV and vascular hypertrophy.
Experimental Animals and Genetic Crosses
Thirteen SHRSP and 13 WKY (6 males and 7 females of each strain) were obtained from colonies maintained at the University of Michigan, which had obtained their breeding stock from the National Institutes of Health (personal communication, Dr D.F. Bohr, Department of Physiology, University of Michigan). These rats were mated brother×sister in Glasgow to provide an SHRSP and a WKY colony as previously described.10 Detailed breeding protocols of two reciprocal crosses have been previously published.11 In brief, for the two reciprocal crosses, 1 male SHRSP was mated with 2 WKY females (cross 1) and 1 male WKY was mated with 2 SHRSP females (cross 2). From the F1 rats of each cross, 3 males and 6 females were brother×sister–mated to generate F2 rats (57 in cross 1 with a male-to-female ratio of 28:29 and 83 in cross 2 with a male-to-female ratio of 37:46). All rats were housed under controlled conditions of temperature (21°C) and light (12-hour light/dark cycle; 7 am to 7 pm) and maintained on normal rat chow (rat and mouse No. 1 maintenance diet, Special Diet Services) and water ad libitum. The pups were weaned and sexed after 3 weeks and housed according to sibling group and sex thereafter.
The Dataquest IV telemetry system (Data Sciences International) was used for measurement of systolic and diastolic BP, mean arterial pressure, HR, and motor activity as previously described.11 In brief, the monitoring system consisted of a transmitter (radiofrequency transducer model TA11PA), receiver panel, consolidation matrix, and personal computer with accompanying software. Rats at 16 weeks of age were anesthetized with halothane, and the flexible catheter of the transmitter was surgically secured in the abdominal aorta just below the renal arteries. The transmitter was sutured to the abdominal wall. The rats were housed in individual cages after the operation, and each cage was placed over a receiver panel connected to the personal computer for data acquisition. The rats were unrestrained and free to move within their cages. Preliminary experiments had shown that BP and HR took up to 12 days postoperatively to stabilize. Therefore, experimental observations collected from days 12 to 16 after surgery were designated “baseline hemodynamic measurements.” Published data suggested that salt-loaded BP may be used as a separate phenotype for genetics studies.3 4 On the evening of day 16, the rats received 1% (wt/vol) NaCl in their drinking water, and this procedure was continued until day 28 when the rats were euthanitized. Measurements collected between days 25 and 28 constituted “hemodynamic measurements on 1% NaCl” or “salt-loaded measures.” Systolic and diastolic BP, HR, and activity were calculated by Dataquest software. Mean values were calculated for 60-minute intervals and exported from the Dataquest program in ASCII format.
The experiments were approved by the Home Office according to regulations regarding experiments on animals in the United Kingdom. These regulations meet all the requirements of the American Physiological Society.
Evaluation of Cardiac Hypertrophy
Immediately after exsanguination the thorax was opened, and the heart was removed, blotted with tissue paper, and weighed. Both atria and the right ventricle were then removed, and the LV and septum were weighed. The ratios of heart weight to body weight and of LV plus septum weight to body weight were determined.
Preparation of Nuclei and Flow-Cytometric Analysis
Vascular smooth muscle cells were obtained from enzymatically digested rat aortas as previously described.12 13 From each aorta, 105 primary vascular smooth muscle cells were used for flow cytometry. The cells were resuspended in phosphate buffer (in mmol/L: 170 NaCl, 3.4 KCl, and 10 Na2HPO4) to a final concentration of 106 cells·mL−1. In sequence, 100 μL of this suspension was treated with 450 μL solution A (30 μg/mL trypsin, pH 7.6) for 10 minutes at room temperature (≈25°C), 375 μL of solution B (0.5 mg/mL trypsin inhibitor and 0.1 mg/mL RNAse, pH 7.6) for another 10-minute incubation at room temperature, and 375 μL of solution C (0.4 mg/mL propidium iodide and 1.1 mg/mL spermine tetrahydrochloride, pH 7.6), which was added directly; this was incubated in the dark at 0°C until analysis. Human peripheral blood lymphocytes were treated as described above to provide a diploid profile for DNA peak standardization. DNA flow cytometry was carried out with a fluorescence-activated cell scanner (Becton-Dickinson UK Ltd) with a 15-mW argon/air-cooled laser and an emission wavelength of 488 nm. Analysis of DNA profiles was performed with the LYSYS software package (Becton-Dickinson).
Genetic Markers and Genotyping
The molecular markers used in the current study consisted of microsatellite polymorphisms typed by PCR. The PCR primers for typing the microsatellite markers by the method of Jacob et al8 were obtained from Research Genetics.Other PCR primers were purchased from Genosys, synthesized in house from previously published sequences,7 8 9 or obtained from The Wellcome Trust Centre for Human Genetics in Oxford (35 primers from Dr Mark Lathrop).
Genotyping was done by PCR amplification of DNA in the region of the microsatellites with a microtiter plate–type apparatus (MJ Research). The reaction volume was 20 μL. Final concentrations were as follows (in mmol/L unless noted otherwise): 45 Tris, pH 8.8; 11 (NH4)2SO4, pH 8.8; 1 MgCl2; 6.7 β-mercaptoethanol; 4.5 μmol/L EDTA; and 25 μmol/L each dATP, dCTP, dGTP, and dTTP. Fifty nanograms of genomic DNA, 0.25 μmol/L of each primer, and 0.4 U Taq polymerase (Promega) were used. The PCR program was as follows: 4 minutes at 94°C and 35 cycles of 1 minute at 94°C, 1 minute at 55°C or 60°C, and 30 seconds at 72°C. For some primers a modified “touchdown” protocol was used, which involved a 0.5°C reduction in annealing temperature during the initial cycles (5°C drop). The final annealing temperature was then used for the last 30 cycles. PCR products were separated by electrophoresis on standard denaturing sequencing gels and transferred to nylon membranes. The membranes were hybridized with a radiolabeled ([α-32P]dCTP) primer at the terminal transferase (Promega). Autoradiographs were independently scored by two investigators, who were unaware of the rats' phenotypes.
Genetic Linkage Analysis
Genetic markers were mapped relative to each other by using the mapmaker/exp 3.0 computer package with an error detection procedure.14 15 Genetic distances were calculated with the Haldane mapping function. QTLs affecting a given phenotype were mapped relative to the genetic markers by using the mapmaker/qtl 1.1 computer package obtained from Dr Eric Lander (Whitehead Institute, Cambridge, Mass). This program calculates the most likely phenotypic effect of having the ss (SHRSP homozygotes) or ww (WKY homozygotes) genotype at a putative QTL and then calculates an LOD score that reflects the strength of the evidence for existence of the QTL and the proportion of the phenotypic variance explained thereby.16 To correct for the effects of multiple-hypothesis testing, it has been suggested that stringent thresholds are required for mapping loci that underlie complex traits, with LOD scores of ≈3 to 3.3 required to establish a significant linkage.6 17 These scores correspond to probability values of ≤10−4. Phenotypic comparisons for different genotypes were performed by using a one-way ANOVA with a conservative significance level of P<.01. In addition, a stepwise regression procedure was used to assess QTL effects while controlling for possible confounding and covariate effects.18 Some QTLs showed possible sex specificity. In these instances we conducted a formal test for the difference in locus effects between sexes. This was accomplished by converting the LOD scores for the whole group, males, and females to likelihood ratios. The computed likelihood-ratio statistic has a χ2 distribution with one degree of freedom. A value >3.85 suggests that the sex difference in the LOD score is significant at the .05 level.
Certain phenotypes were clearly not normally distributed. Values for vascular smooth muscle polyploidy and motor activity were positively skewed; therefore, the values for these parameters were logarithmically transformed before inclusion as dependent variables. The distributions of body, whole heart, and LV weights were strongly bimodal (due to sex differences), but the distributions for each sex separately were acceptably normal, so these three phenotypes were analyzed separately by sex.
Radiotelemetry BP measurements were obtained for 13 SHRSP (male/female=7:6), 12 WKY (male/female=6:6), 22 F1 (male/female=13:9), and 140 F2 (male/female=65:75) rats at baseline and after salt loading. We were unable to measure salt-loaded BP in the SHRSP males because of the very high mortality in this group. Therefore, a precise estimate of the degree of genetic determination of salt-loaded BP subphenotype was not possible in the F2 males. The degree of genetic determination of baseline BP and salt-loaded BP subphenotypes in the female F2 cohort is shown in Table 1⇓.
A total of 603 microsatellite markers were screened, and of these, 181 were found to be polymorphic between SHRSP and WKY. This gives a 30% polymorphism rate. Using these markers and the mapmaker/qtl 1.1 computer program, we identified a QTL on rat chromosome 2 with a significant LOD score of 3.3 for baseline systolic BP and a LOD score of 3.4 for baseline diastolic BP (Fig 1⇓). These two QTLs accounted for 12.9% and 13.8% of the variance in systolic and diastolic BP, respectively. The same analysis for salt-loaded BPs showed “suggestive” QTLs in the same chromosome region, with LOD scores of 2.9 and 2.7 that accounted for 12.3% and 12.0% of the variance in salt-loaded systolic and diastolic BP, respectively. We then performed the same analysis for male F2 hybrids only. This analysis revealed a QTL in the same region of rat chromosome 2, with LOD scores of 3.65 and 3.5 for baseline systolic and diastolic BP, which accounted for 25% and 24% of the respective variance in BP (Fig 2⇓). Salt-loaded systolic and diastolic BPs in male F2 cohorts gave LOD scores of 3.4 and 3.2, accounting for 31% and 26% of the respective variance in BP (Fig 3⇓). Because D2Mit6 is the closest marker linked to the QTL described above, we examined the influence of this marker on a number of phenotypes with a standard one-way ANOVA. Table 2⇓ shows that the locus characterized by the D2Mit6 marker had a strong effect on baseline systolic and diastolic BPs and a lesser but still significant effect on salt-loaded systolic and diastolic BPs in the combined data set (males and females analyzed together; N=140). The same analysis for the male F2 hybrids alone shows a significant effect of the locus characterized by the D2Mit6 marker on all four subphenotypes of BP. However, this marker does not appear to significantly influence other phenotypes, such as LV weight to body weight ratio or vascular hypertrophy (Table 2⇓). Moreover, the D2Mit6 locus affects BP in a recessive manner, since homozygous ww and heterozygous ws rats have equivalent BPs, whereas homozygous ss rats have significantly elevated BPs (Table 2⇓).
Further analysis of male F2 hybrids revealed a potential second QTL on rat chromosome 2, which was localized between markers D2Mit14 and D2Mgh12. The LOD scores for this QTL were as follows: 2.0, 2.5, 2.3, and 3.1 for baseline systolic, baseline diastolic, salt-loaded systolic, and salt-loaded diastolic BPs, respectively. The two polymorphic markers were on the downslope of the LOD plot, and therefore a one-way ANOVA would not have been appropriate to verify these results. The map distance between the significant QTL proximal to D2Mit6 and the “suggestive” QTL between markers D2Mit14 and D2Mgh12 was calculated at ≈73 cM (Fig 2⇑).
Data analysis for male F2 hybrids also revealed a QTL on rat chromosome 3, with D3Mgh16 being the closest marker to this QTL. BP subphenotypes that showed significant LOD scores in this region were baseline and salt-loaded pulse pressures (LOD scores of 4.2 and 5.6, which contributed 32.2% and 39.8% of the variance in pulse pressure, respectively) and salt-loaded systolic and diastolic BPs (LOD scores of 4.4 and 3.3, which contributed 39.6% and 36.7% of the variance in BP, respectively; Fig 4⇓). These results were confirmed by one-way ANOVA, with baseline pulse pressure being of borderline significance according to the stringent criteria we adopted (Table 3⇓). Similar to D2Mit6, the locus close to D3Mgh16 affected BP phenotypes in a recessive manner.
Two QTLs (D2Mgh12 and D3Mgh16) showed possible sex specificity. We therefore conducted a formal test for the difference in locus effect between the sexes by using a simple likelihood-ratio test. The results are shown in Table 4⇓. For each QTL localized close to D2Mgh12 and D3Mgh16, the likelihood-ratio statistic was significant, thus confirming the sex difference in LOD scores.
Furthermore, we detected a QTL on rat chromosome 14 for the ratio of LV weight to body weight. This QTL was localized between markers D14Mgh3 and R58, which are ≈12.3 cM apart. Similar to the “suggestive” QTL on rat chromosome 2, the two polymorphic markers are on the downslope of the LOD plot and again a one-way ANOVA is not appropriate to verify these results. Nevertheless, LOD scores for this phenotype were 3.7 for F2 males and 3.1 for F2 males and females analyzed together, which accounted for 32.3% and 12.3%, respectively, of the variance in LV weight to body weight ratio.
No other QTLs were detected in our genome scan. Specifically, we detected no QTLs for vascular smooth muscle polyploidy (a marker of vascular hypertrophy), HR, or motor activity. We therefore performed a stepwise regression procedure to assess the contribution of individual QTLs, as detected by linkage analysis, and the putative Y chromosome locus to the BP phenotypes under study, as previously reported by ourselves,11 Turner et al,19 and Ely et al.20 The results of stepwise regression with baseline systolic BP as the dependent variable are shown in Table 5⇓. The SHRSP Y-chromosome effect, D2Mit6 recessive effect, LV weight to body weight ratio, and vascular smooth muscle polyploidy markedly but independently influenced baseline systolic BP. Together they accounted for 43% of the variance in total baseline systolic BP. We also performed a stepwise regression with salt-loaded pulse pressure as the dependent variable (Table 5⇓). The D3Mgh16 recessive effect, vascular smooth muscle polyploidy, the SHRSP Y-chromosome effect, and LV weight to body weight ratio markedly but independently influenced salt-loaded pulse pressure. Similar to the previous analysis, together they accounted for 43.2% of the total variance in salt-loaded pulse pressure.
The novel observation of the current study is the identification of possible sex-specific loci on rat chromosomes 2 and 3 that influence BP. A genetic map for rat chromosome 2 includes several candidate genes that have been previously studied in crosses of DSS and WKY or MNS rats.21 22 These studies identified a BP QTL localized between the Na+,K+-ATPase α1 isoform and calmodulin-dependent protein kinase IIΔ loci with an LOD score of 5.66. This QTL accounted for 9.2% of the total variance in systolic BP and ≈26% of the genetic variance. Other data suggested the presence of a second BP QTL on chromosome 2 near an angiotensin type 1B receptor gene in the F2 (MNS×DSS) population. This region has also been implicated in an F2 population derived from a cross of Lyon-hypertensive and Lyon-normotensive rats, in which systolic and pulse pressures were significantly correlated with a CPB gene.23 CPB is located very close (≈0.8 cM) to the angiotensin type 1B receptor gene.
Previous studies used F2 males only for linkage analyses. The current study used male and female F2 cohorts to enable us to identify sex-specific QTLs if they exist. First, we identified a baseline BP QTL with its peak close to D2Mit6. This anonymous marker lies ≈9.8 cM from the CPB gene and likely corresponds to the previously identified QTL in this region. Second, we identified a “suggestive” QTL on rat chromosome 2, localized ≈73 cM away from D2Mit6, which contributed to salt-loaded BP. This QTL appears to be sex specific, being present in the male F2 cohort only. This sex specificity was confirmed by a significant likelihood-ratio test, which confirmed the sex difference in the LOD scores. The QTL is localized between two anonymous markers, D2Mgh12 and D2Mit14, with the latter being ≈13 cM from guanylyl cyclase A, a region implicated in previous studies.21 22 Furthermore, we identified a second sex-specific BP QTL on rat chromosome 3 in the region of the anonymous marker D3Mgh16. Again, this QTL was present in male F2 rats only and showed a significant likelihood-ratio test of the sex difference in LOD scores. It is of interest that Nara et al24 found a significant linkage of D3Mgh16 with basal systolic BP in male and female SHRSP×WKY (Izumo) crosses. The difference between the two studies might be related to different methods of BP measurement or to the previously reported genetic difference between WKY and SHRSP strains originating from the US National Institutes of Health and those from colonies in Japan.25 The current study is the first to report the use of radiotelemetry for phenotyping an entire F2 cohort in a linkage study. It is likely that such high-fidelity phenotyping, in which the animals are free from catheters, tethers, and stress from human contact, may affect the final results of QTL mapping.
There is no other study that has reported possible sex specificity for BP QTLs. Melo et al26 recently identified two sex-specific QTLs that control alcohol preference in mice: a male-specific locus on mouse chromosome 2 and a female-specific locus on mouse chromosome 11. Two hypotheses were advanced to explain these results: first, the existence of an X-linked locus with complementary activity to a female-specific locus and second, genomic imprinting. Our results may have resulted from interaction between the Y-linked locus and other BP QTLs. However, the stepwise regression procedures carried out for baseline and salt-loaded BP phenotypes did not reveal such an interaction. Instead, the putative SHRSP Y-chromosome effect and the D2Mit6 or D3Mgh16 recessive effects showed a significant but independent influence on baseline and salt-loaded BP. Genomic imprinting would require the loci to be present on chromosomes 3 and 2, with the only functional copy of each locus having been inherited from the father. We previously considered the possibility that genomic imprinting might have mimicked Y-chromosome linkage.11 Parental imprinting is characterized by a phenotypic difference of the same gene, depending on whether this gene is inherited from the male or female parent.27 For example, the c-myc transgene shows different methylation patterns; only those offspring that inherit the transgene from their father can express the gene.27 By analogy, it is possible that an autosomal hypertensive allele is active only if inherited from the male parent. To prove this hypothesis, it would be necessary to demonstrate that F2 females with an SHRSP grandfather have higher BPs than do those with a WKY grandfather. Our previous detailed analysis of baseline telemetry BPs did not show such a difference.11 It is of interest that F2 females with an SHRSP grandfather had significantly higher salt-loaded pressures than did F2 females with a WKY grandfather.11 It has also been shown that some BP QTLs cosegregate predominantly with BP after exposure to excess dietary salt.3 4 5 18 Therefore, it may be possible that parental imprinting is relevant only to those loci that influence salt-loaded BP.
In addition to BP QTLs, we also identified a QTL on rat chromosome 14 for LV hypertrophy. In view of the borderline LOD score of 3.1 in this region, it should be considered as a “suggestive” linkage only.17 A recent study of recombinant inbred strains showed significant linkage of the microsatellite marker within the dopamine 1A receptor on chromosome 17 with LV heart weight adjusted for body weight.28 The relationship between dopamine 1A receptor genotype and LV mass was independent of BP, but the SHR allele contributed to lower LV mass in the recombinant inbred strains.28 The current study has revealed a second putative QTL that regulates cardiac mass. It is likely that other “susceptibility” loci for cardiovascular complications of hypertension might be identified by using similar strategies. Brown et al2 demonstrated that renal disease susceptibility is at least partially under genetic control independent from susceptibility to hypertension itself.
It is recognized that the identification by linkage analysis of large chromosome regions containing QTLs is only the first step in the ultimate positional cloning of the QTLs.29 The next step is production of congenic strains and substrains with progressively smaller chromosomal regions and the final task of cloning candidate genes.29 Identification of sex-specific QTLs on rat chromosomes 2 and 3 and the putative QTL that regulates LV mass will lead to new congenic strategies and, ultimately, positional cloning of the BP QTLs.
Selected Abbreviations and Acronyms
|DSS||=||Dahl salt-sensitive rats|
|LOD||=||logarithm of the odds|
|PCR||=||polymerase chain reaction|
|QTL||=||quantitative trait locus/loci|
|SPSHR||=||stroke-prone spontaneously hypertensive rats|
This work was supported by British Heart Foundation grants PG 92100, PG 95123, and FS 93025 to A.F.D. and by the European Community Biomed Program (EURHYPGEN Concerted Action). A.F.D. is a British Heart Foundation Senior Research Fellow. The authors thank Drs Mark Lathrop and Dominique Gauguier for microsatellite markers and their help in the development of new methods. We also thank Angela McKay for typing the manuscript.
- Received July 22, 1996.
- Revision received July 29, 1996.
- Accepted August 15, 1996.
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