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Physiol. Genomics 26: 172-179, 2006. First published May 23, 2006; doi:10.1152/physiolgenomics.00285.2005
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Received 18 November 2005; accepted in final form 11 April 2006.
Physiological Genomics 26:172-179 (2006)
1094-8341/06 $8.00 © 2006 American Physiological Society

Sex-specific QTLs and interacting loci underlie salt-sensitive hypertension and target organ complications in Dahl S/jrHS hypertensive rats

Victoria L. M. Herrera, Aristides Tsikoudakis, Lorenz R. B. Ponce, Yuichi Matsubara and Nelson Ruiz-Opazo

Section of Molecular Medicine, Department of Medicine, and Whitaker Cardiovascular Institute, Boston University School of Medicine, Boston, Massachusetts


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 GRANTS
 REFERENCES
 
Sex-specific differences in polygenic (essential) hypertension are commonly attributed to the role of sex steroid hormone-receptor systems attenuating sex-common disease mechanisms in premenopausal women. However, emerging observations indicate sex-specific genetic susceptibility in various traits, thus requiring systematic study. Here we report a comparative analysis of independent total genome scans for salt-sensitive hypertension susceptibility quantitative trait loci (QTLs) in male and female F2 [Dahl R/jrHS x S/jrHS] intercross rats exposed to high-salt (8% NaCl) rat diets. Hypertension was phenotyped with three quantitative traits: blood pressure (BP) elevation associated with increased hypertensive renal disease [glomerular injury score (GIS)] and increased cardiac mass [relative heart weight (RHW)] obtained 8–12 wk after high-salt challenge; 24-h nonstress, telemetric BP measurements were used. Although sex-common QTLs were detected for BP [chromosome (chr) 1–144.3 Mbp; chr 1–208.8 Mbp], GIS (chr 1–208.8 Mbp), and cardiac mass (chr 5–150.3 Mbp), most QTLs across the three phenotypes studied are gender specific as follows: female QTLs for BP (chr 2–106.7 Mbp, chr 2–181.7 Mbp, chr 5–113.9 Mbp, chr 5–146.7 Mbp, chr 12–12.8 Mbp), GIS (chr 15–59.6 Mbp), and RHW (chr 2–31.5 Mbp, chr 5–154.7 Mbp, chr 5–110.9 Mbp); male QTLs for BP (chr 2–196.7 Mbp, chr 11–48.0 Mbp, chr 20–35.7 Mbp), GIS (chr 6–3.3 Mbp, chr 20–40.7 Mbp), and RHW (chr 6–3.3 Mbp, chr 20–40.7 Mbp). Furthermore, interacting loci with significant linkage were detected only in female F2 intercross rats for BP and hypertensive renal disease. Comparative analyses revealed concordance of BP QTL peaks with previously reported rat model and human hypertension susceptibility genes and with BP QTLs in previous Dahl S-derived F2 intercross studies and also suggest strain-specific genetic modifiers of sex-specific determinants. Altogether, the data provide key experimental bases for sex-specific investigation of mechanisms and intervention and prevention strategies for polygenic hypertension in humans.

sex-specific susceptibility; genetics; blood pressure; renal disease; relative heart weight; quantitative trait locus


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 GRANTS
 REFERENCES
 
POLYGENIC (ESSENTIAL) HYPERTENSION is a leading risk factor for heart disease, stroke, and renal failure (10, 14, 16, 22, 33). Despite increasing efforts to decipher its etiology, the genetic determinants of susceptibility to hypertension and its target organ complications remain to be fully elucidated. To add to the complexity of elucidating molecular genetic mechanisms of this chronic condition, differences between men and premenopausal women in pathophysiology, risks, and treatment have been reported (9, 28, 31, 41). While documenting progress, cumulative reviews (2, 26, 30, 40) chronicle the emerging complexity and the present incomplete understanding of mechanisms underlying sex-specific differences, thus emphasizing the need for continuing investigation of this issue. The logical premise that the attenuated hypertension phenotype in females is solely due to the modulation of identical sex-common determinants by sex steroid hormones in premenopausal women needs to be tested. Testing the hypothesis that sex-specific hypertension susceptibility involves sex-specific genetic determinants in addition to, and distinct from, sex steroid hormone-mediated attenuation of sex-common determinants is facilitated in valid animal model genetic studies, because animal models provide a robust experimental platform to investigate complex biological traits by controlling multiple confounders. The inbred Dahl salt-sensitive (S) and salt-resistant (R) hypertensive rat strains comprise a well-validated hypertensive and control model of polygenic (essential) hypertension (37). Furthermore, in the Dahl S rat strain, hypertension is less in premenopausal females compared with age-matched males (5, 19, 38), recapitulating the greater prevalence of essential hypertension in males compared with age-matched premenopausal females (9, 28, 41). This validates the Dahl S rat strain as a model of human sex-specific genetic susceptibility to hypertension.

To investigate sex-specific determinants of genetic susceptibility, valid comparative analyses of male and female genetic cohorts require the following study components that, although not feasible in humans, are experimentally robust in animal model studies: ascertainment of identical environmental factors such as diet, housing, and handling; elimination of genetic background heterogeneity by deriving male and female F2 intercross cohorts from identical parental strains; and phenotype characterization with identical parameters. Comparative analysis using a total genome scan strategy provides the simultaneous investigation of multiple quantitative trait loci (QTLs) of complex traits acting as major genes or interacting genes contributing to said susceptibility without a priori hypotheses. Aside from the identification of QTLs, observations from a total genome scan can address whether sex-specific hypertension susceptibility is rooted in a quantitative paradigm, attenuation of identical genetic determinants between sexes; is rooted in a qualitative paradigm, different sets of QTLs and/or interacting loci between sexes; or involves both. The projected significance cannot be overemphasized, because a different set of QTLs would imply different molecular targets for detection, intervention, and prevention for men and women, which presently is not the norm.

Here we report a comparative total genome scan for QTLs underlying salt-sensitive hypertension disease course characterized by blood pressure (BP) elevation and hypertensive target organ complications in F2 intercross male and female populations derived from Dahl salt-resistant (Dahl R/jrHS) and Dahl salt-sensitive (Dahl S/jrHS) hypertensive inbred rat lines. Established quantitative phenotype parameters were used for the comparative total genome scan: 1) systolic blood pressure (SBP) measured by 24-h nonstress radiotelemetry, 2) hypertensive renal disease score measured as glomerular injury score (GIS), and 3) cardiac mass measured as relative heart weight-to-body weight ratio (RHW). Interestingly, most QTLs detected across the three phenotypes were sex specific, and interacting loci with significant linkage were detected only in the female F2 [R x S] intercross cohort and not in males. Altogether, the data support the dual hypothesis that although there are a few sex-common QTLs that support a quantitative paradigm of estrogen-mediated attenuation, the majority of QTLs and interacting loci detected are sex specific, supporting a qualitative paradigm of distinct genetic determinants of hypertension susceptibility between sexes.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 GRANTS
 REFERENCES
 
Genetic crosses.
Inbred Dahl S/jrHS and Dahl R/jrHS rats were obtained from Harlan (Indianapolis, IN). Parental strains (Dahl R/jrHS female x Dahl S/jrHS male) were crossed to produce F1 progeny. The F2 subjects were derived from brother-to-sister mating of F1 hybrids to produce the F2 male (n = 106) and F2 female (n = 102) segregating populations.

Phenotypic characterization.
All animal procedures were performed in accordance with institutional guidelines and have been approved by the Boston University Animal Care and Use Committee. Animals were maintained on a LabDiet 5001 rodent chow (Harlan Teklad, Madison, WI) containing 0.4% NaCl from weaning until the high-salt diet began at 12 wk of age. Food pellets and water were made available ad libitum. BP was measured essentially as described previously (19) with intra-aortic abdominal radiotelemetric implants (Datascience) obtaining nonstressed BP measurements, taking the average over 10 s every 5 min for 24 h (19). SBP, diastolic blood pressure (DBP), and mean arterial pressure (MAP) were obtained along with heart rate and activity. The protocol for parental and F2 intercross rats was as follows: implant surgery at 10 wk of age (only rats with no postoperative complications were used); after 12 days, baseline BP levels were obtained. The high-salt (8% NaCl) challenge was initiated at 12 wk of age and maintained for 8 and 12 wk for male and female rats, respectively; a longer high-salt challenge was necessary for females to attain a similar mean BP because BP in females is lower. BP values used for phenotype are the averages obtained in the final week of the salt loading from a 24-h recording during a no-entry day (Sunday) ascertaining nonstress BP. A BP reading was obtained every 10 min; the 24-h average was selected to account for circadian rhythm BP changes and was observed to be the most robust phenotype measure for BP-genotype linkage. Heart and body weights were obtained at the time of death (20 wk of age for males; 24 wk of age for females). Renal pathology was assessed essentially as described previously (35). After death, the kidneys were removed, fixed in 4% paraformaldehyde, and processed for histology, and renal pathology was quantified as described previously (19). All glomeruli in one frontal renal section were analyzed in a blind manner for degree of glomerulosclerosis and mesangial matrix expansion. Glomerulosclerosis was defined as disappearance of cellular elements from the tuft, collapse of capillary lumen, and folding of the glomerular basement membrane with entrapment of amorphous material. Mesangial matrix expansion was defined by the presence of increased amounts of periodic acid Schiff-positive material in the mesangial region. Renal pathology grades were I, 25% involvement of glomerulus with pathology; II, 50% involvement; III, 75% involvement; and IV, 100% involvement. The extent of injury for each renal section was calculated as the GIS = (1 x % grade I) + (2 x % grade II) + (3 x % grade III) + (4 x % grade IV), increasing with worse injury represented by glomerulosclerosis and mesangial matrix expansion (35). Statistical analyses performed were one-way ANOVA, all pairwise multiple comparisons with Tukey test, regression analysis, and t-test (SigmaStat).

Intercross linkage analysis.
Genotyping was done by the National Heart, Lung, and Blood Institute (NHLBI) Mammalian Genotyping Service at the Center for Medical Genetics, Marshfield Medical Research Foundation (Marshfield, WI). We used 121 microsatellite markers informative for our F2 [R x S] intercross with an average density of 12.4 cM. In addition, both cohorts were genotyped with EA4, a previously described single-strand conformation polymorphism marker for the Dear locus detecting the S44P/M74T (Dahl R) and S44/M74 (Dahl S) gene variants (25). Distributions were analyzed for normality; data transformations were done, and data sets that passed Kolmogorov-Smirnov normality testing (SigmaStat) were used for linkage analysis. QTL analysis was performed with log [SBP], heart weight/body weight (RHW), and GIS as quantitative traits. In both male and female F2 intercross populations renal disease (GIS) showed no significant relationship with BP on Pearson product moment correlation analysis (F2 male cohort: correlation coefficient = –0.125, P > 0.2; F2 female cohort: correlation coefficient = 0.081, P > 0.4); thus QTL analysis for renal disease was performed without adjustment for BP. QTL analysis for RHW in both male and female F2 cohorts were done with RHW adjusted for SBP because these traits were significantly correlated (F2 male cohort: correlation coefficient = 0.53, P < 10–8; F2 female cohort: correlation coefficient = 0.43, P < 10–5). Linkage maps, marker regression, and composite interval mapping were done with the Map Manager QTXb19 (MMQTXb19) program for Windows (29), which generates a likelihood ratio statistic (LRS) as a measure of the significance of a possible QTL. Genetic distances were calculated with Kosambi mapping function (genetic distances are expressed in cM). Critical significance values (LRS values) for interval mapping were determined by a permutation test (2,000 permutations at 10-cM interval) on our male and female cohorts with Kosambi mapping function and a free regression model. This permutation analysis revealed the minimum values for suggestive linkage LRS = 8.7 [logarithm of the odds score (LOD) 1.9]: for significant linkage, LRS = 14.9 (LOD 3.2); for highly significant linkage, LRS = 22.5 (LOD 4.9). LRS 4.6 delineates the LOD 1 support interval. Regression analysis using a free model fit as well as constrained additive, dominant, and recessive models were applied. Data are presented for the free model fit because this analysis fits separate regression coefficients for both additive and dominance components (QTX Map Manager, MMQTXb19).

The confidence interval for a QTL location was estimated by bootstrap resampling method in which a histogram single peak delineates the QTL and peak widths define the confidence interval for the QTL. Histograms that show more than one peak warn that the position for the QTL is not well defined or that there may be multiple linked QTLs (QTX Map Manager).

Interaction analysis.
Interaction analysis was done with the Map Manager QTXb19 program, applying a two-stage test paradigm for determination of interaction in which the pair of loci must pass two tests to be reported as having a significant interaction effect. First, the significance of the total effect of the two loci must be <0.00001 and second, the pairs of loci must exhibit a P value <0.01 for the interaction effect.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
GRANTS
 REFERENCES
 
Phenotype characteristics in parental strains.
To investigate sex-specific hypertension susceptibilities, we ascertained that the Dahl salt-sensitive hypertensive (S) and Dahl salt-resistant normotensive (R) rat strains comply with a priori dual prerequisites: contrasting hypertension phenotypes in parental strains and significantly less hypertension in females. We also focused our genetic analysis on evaluating hypertension as a disease—BP elevation associated with end-organ disease—and not just BP elevation after a 10-day high-salt diet challenge. Phenotype parameters were thus measured after 8 (male)- and 12 (female)-wk high-salt challenge that began at 12 wk of age, coinciding with significant BP elevation and expected target organ complications of hypertensive renal disease (GIS) and increased cardiac mass (RHW). As shown in Table 1, radiotelemetric 24-h average BP, RHW, and GIS are significantly higher in Dahl S compared with Dahl R rat strains in both males (P < 0.001) and females (P < 0.001), ascertaining contrasting hypertension disease phenotypes in parental strains. RHW values were not significantly different (P > 0.7, Table 1) between male and female F2 cohorts. However, the F2 male population showed higher mean BPs and GIS compared with the F2 female population (P < 0.001, Table 1) despite the increased duration of the high-salt challenge imposed on the female rats (8 and 12 wk for male and female rats, respectively), recapitulating the relative hypertension resistance in females. The 12-wk time point was used for females to optimize the power of phenotype-genotype analysis.


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  		Table 1. Mean blood pressure, relative heart weight, and glomerular injury score in Dahl S, Dahl R, and F2 cohorts

 
Most QTLs detected are sex specific.
To identify putative QTLs influencing BP, GIS, and RHW in male and female Dahl rats, we compared independent QTL analysis on F2 [R x S] intercross male and female cohorts phenotyped for BP, GIS, and RHW. In both male and female F2 intercross populations, GIS and RHW exhibited normal distribution. SBP data were transformed (log [SBP]) to pass normality testing. QTL analysis was similar among SBP, DBP, and MAP, with SBP giving the most robust results and hence presented here.

One hundred and six F2 male hybrids were genotyped at 121 markers informative for Dahl S and Dahl R strains. With a free regression model, two QTLs influencing BP were detected with significant to highly significant linkage (Table 2) on chromosome 1, BP-m1 (LOD = 5.1) and BP-m2 (LOD = 3.5), and three BP QTLs with suggestive linkage were detected: one each on chromosome 2 (BP-m4; LOD = 1.9), chromosome 11 (BP-m5; LOD = 1.9), and chromosome 20 (BP-m3; LOD = 2.4).1 Linkage analysis of GIS detected two GIS QTLs with significant linkage, on chromosome 2 GIS-m1 (LOD = 3.5) and on chromosome 11 GIS-m2 (LOD = 3.4), and three GIS QTLs with suggestive linkage, one on chromosome 18 (GIS-m3; LOD = 2.9), one on chromosome 5 (GIS-m4; LOD = 2.7), and one on chromosome 1 (GIS-m5; LOD = 2.2). Linkage analysis of RHW detected two RHW QTLs with significant linkage, one on chromosome 20 (RHW-m1; LOD = 3.6) and one on chromosome 5 (RHW-m2; LOD = 3.4), and one RHW QTL with suggestive linkage on chromosome 6 (RHW-m3; LOD = 2.6). Analysis of Dahl S allele specific effects reveals differential effects of S alleles on trait values. The S allele increases trait values in most detected QTLs, except for BP-m5, GIS-m1, and RHW-m3, in which the S allele contributes to a decrease in the trait values, respectively (Table 2).


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  		Table 2. QTLs with significant and suggestive linkage for SBP, GIS, and RHW in F2[R x S] intercross male rats

 
For the female total genome scan, 102 F2 female hybrids were genotyped with the same markers used for the F2 male cohort. With a free regression model, we detected four BP QTLs with significant to highly significant linkage (Table 3): two on chromosome 5, BP-f1 (LOD = 4.3) and BP-f2 (LOD = 3.9); one on chromosome 12, BP-f3 (LOD = 3.5); and one on chromosome 2, BP-f4 (LOD = 3.5). We detected QTLs with suggestive linkage for BP on chromosome 2 (BP-f5; LOD = 2.1) and on chromosome 1 (BP-f6; LOD = 2.1). In contrast to the male cohort, linkage analysis for hypertensive renal disease and cardiac mass did not reveal any QTLs with significant linkage, only QTLs with suggestive linkage (Table 3), perhaps attributable to the lower level of BP elevation despite the longer duration of high-salt diet challenge in the F2 intercross female cohort. We detected a single QTL with suggestive linkage for GIS on chromosome 15, GIS-f1 (LOD = 2.5). We detected three QTLs with suggestive linkage for RHW: two QTLs on chromosome 5, RHW-f1 (LOD = 2.9) and RHW-f3 (LOD = 2.0), and one on chromosome 2, RHW-f2 (LOD = 2.3). In contrast to varied S allele effects in the male F2 intercross cohort, QTL analysis in F2 intercross females showed that the QTL-specific S alleles increase trait values for all phenotypes analyzed (Table 3).


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  		Table 3. QTLs with significant and suggestive linkage for SBP, GIS, and RHW in F2 [R x S] intercross female rats

 
Interaction analysis detects significant interacting loci only in females.
Putative interactive loci were investigated by using a two-stage test that determines that only pairs of loci that show a P value <0.00001 (LOD > 8.0) for the total effect and a P value <0.01 (LOD > 4.1) for the interaction effect are considered positive for interaction (29). Our analysis revealed no gene-gene interaction in the male F2-intercross cohort. In contrast, several interacting loci were detected in the female F2 intercross cohort in two phenotypes studied that surpassed the threshold criteria for gene interaction (Table 4). One pair of interacting loci for renal disease score was detected on GIS-f1 with a locus on chromosome 1 marked by D1Mgh11 (ixGIS-f1). Three pairs of interacting loci for BP were detected. Interestingly, all three interacting loci for BP involve a major gene/BP QTL with significant linkage in females, BP-f2 QTL, on chromosome 5. BP-f2 interacts with three independent loci marked by D1Mgh11 (ixBP-f1), D3Rat18 (ixBP-f2), and D14Rat90 (ixBP-f3), respectively (Table 4). A priori, these observations highlight the importance of BP-f2 as a major gene/QTL and as an "anchor" for three interacting loci—all with significant linkage to salt-sensitive hypertension susceptibility. Additionally, we note that interacting loci partners of BP-f2 would not otherwise be detected unless interaction analysis was performed (Table 4).


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  		Table 4. Interaction analysis

 
Comparative analyses of single locus effect QTLs and interacting loci for hypertension susceptibility.
Comparative analysis of single locus effect QTLs and interacting gene loci for hypertension, hypertensive renal disease, and RHW between male and female F2 intercross cohorts (Fig. 1; Tables 2 and 3) revealed that out of 9 single locus effect QTLs with significant linkage, 13 single locus effect QTLs with suggestive linkage, and 4 interacting loci with significant interaction (Tables 2–4), only 7 localize to 3 sex-common susceptibility loci for hypertension disease (Fig. 1). Two of these sex-common susceptibility loci exhibit linkage to BP: 1) QTL peak at chromosome 1, 144.3 Mbp for both BP-m1 with significant linkage and BP-f6 with suggestive linkage, and 2) QTL peak at chromosome 1, 208.2 Mbp for both BP-m2 and ixBP-f1, both with significant linkage (Tables 2 and 3; Fig. 1). Interestingly, this QTL peak also corresponds to GIS in both sexes, although with different linkage degrees: GIS-m5 with suggestive linkage in males and ixGIS-1f with significant linkage. Correspondence of both BP and GIS in both sexes strongly suggests the importance of this QTL in influencing susceptibility to hypertension and renal end-organ disease. A third sex-common susceptibility QTL at chromosome 5, 150.3 Mbp exhibits significant linkage to different phenotype parameters for hypertension disease and RHW in males, RHW-m2, and SBP in females, BP-f1 (Fig. 1).


Figure 1
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  		Fig. 1. Gender-specific quantitative trait loci (QTLs) with significant linkage for blood pressure (BP), glomerular injury score (GIS) and relative heart weight (RHW). QTLs detected on total genome scans are plotted per chromosome according to relative chromosome position in Mbp. Interacting loci are connected by dashed lines and like symbols. ix, Interacting loci; m, male; f, female.

 
Comparative analysis also highlights the detection of identical QTL peaks between suggestive loci and significant loci, albeit for different hypertension disease parameters (Fig. 1). Two involve single locus effect QTLs with suggestive linkage for RHW and with significant linkage for BP: 1) RHW-f1 coincides with BP-f1, and 2) RHW-f3 coincides with BP-f2 (Fig. 1). A third involves like-parameter QTLs: a single locus effect QTL with suggestive linkage for GIS coinciding with a significant interacting locus for GIS, ixGIS-f1. Although corroboration of all QTLs is necessary, coincident peaks of like or related hypertension disease parameters support the likelihood of said suggestive loci as genetic determinants.

QTL peak-focused candidate gene analyses.
Although QTLs detected in total genome scans require corroboration and further narrowing because QTL regions (±1 SD from the peak) invariably span multiple genes, analysis of candidate susceptibility genes following preset criteria is a valid approach to forwarding mechanistic hypotheses. Identification of candidate genes also facilitates the much-needed comparative analyses across previous studies done using different strain-specific informative markers, as well as across species. We limited candidate gene analysis to QTLs with significant linkage or to QTLs that correspond to previously reported hypertension genes exhibiting BP-relevant, functionally significant variants and association/linkage in single candidate gene analyses. We used the following criteria for designating which gene(s) comply as candidate genes: 1) proximity to QTL peaks, defined within a 15-cM range and <1 SD from the QTL peak, 2) functionally significant variants with relevance to BP modulation or salt sensitivity, and 3) previous linkage or association with BP. This analysis detects the following previously reported hypertension susceptibility genes within BP QTLs detected in this total genome scan: {alpha}-adducin (42), {alpha}1Na,K-ATPase (4, 15, 18, 19, 24), bumetanide-sensitive Na,K,2Cl cotransporter (15, 20), dual ANG II/AVP receptor (39), dual ET-1/ANG II receptor (25), and prolylcarboxypeptidase (Prcp) or angiotensinase C (43). Because all these genes were identified based on a candidate gene analysis paradigm of functionally significant variants, corroboration in the present total genome scan reaffirms the likelihood of these genes as hypertension susceptibility genes. Additionally, BP-f2, with significant linkage to high BP in females, spans barttin (Bsnd), which underlies a form of monogenic hypertension, Barter syndrome (23). Although further study is necessary to assign barttin as the hypertension gene, the detection of a QTL for polygenic hypertension at a monogenic hypertension gene locus provides experimental support for the long-hypothesized putative role of a monogenic hypertension gene in polygenic hypertension for the first time.

QTLs common to BP and end-organ complications.
Previous studies have identified distinct QTLs for BP and hypertensive end-organ complications such as renal disease and increased cardiac mass, suggesting differential genetic susceptibility mechanisms. We corroborated this phenomenon in this total genome scan but also detected QTLs that are common to BP and end-organ renal disease and common to both sexes, in males BP-m2 and GIS-m5 and in females ixBP-f1 and ixGIS-f1, with significant interaction contributing to renal disease in females (Fig. 1). Although total genome analysis detects differential linkage modalities—single locus effect QTLs in males and interacting loci in females—the commonality of QTL detection for two hypertension disease parameters, BP and GIS, and the commonality of detection in both male and female F2 intercross cohorts provide strong support for this QTL as a susceptibility locus for hypertension as a disease.

Normotensive genetic backgrounds affect sex specificity of BP QTLs.
Because strain-specific BP QTLs have been detected, suggesting genetic subtypes of hypertension, we performed parallel comparative analyses to gain further insight into sex specificity of hypertension QTLs. We compared QTL peaks detected here with QTL peaks detected in total genome scans of other F2 intercross cohorts sharing a common hypertensive parental strain, the Dahl S rat, but intercrossed with a non-Dahl R normotensive parental strain. Keeping the F2 intercross parental hypertensive strain constant allows for informative discordances. Analyzing QTL peaks rather than the region representing 95% confidence limits (2-LOD or 20-cM region; Ref. 37) places emphasis on the highest likelihood region for the "culprit gene" proven repeatedly in human genetics and facilitates analysis of candidate hypertension genes in humans based on synteny.

Despite different BP phenotyping protocols, comparative analysis reveals two BP QTLs that are common across different Dahl S-derived F2 intercross cohorts located in chromosome 1 (chr 1–131.1 Mbp, chr 1–144.3 Mbp, and chr 1–148.3 Mbp) and chromosome 2 (chr 2–218.9 Mbp and chr 2–208.8 Mbp) (Table 5). Common F2 intercross QTL regions could represent major hypertension susceptibility loci eliciting phenotype regardless of genetic background modifiers, or, alternatively, they could represent QTL clusters (21).


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  		Table 5. Comparative analysis of QTL peaks detected in total genome studies of F2 intercross cohorts derived from inbred Dahl S rats as the hypertensive parental strain

 
More interestingly, three female-specific QTL peaks detected in our F2 [Dahl R x Dahl S] cohort overlap with BP QTL peaks detected in male F2 [non-Dahl R x Dahl S] intercross cohorts: 1) chr 2–105.5 Mbp in male F2 [Dahl S x AS] (7) and BP-f5 at chr 2–107.6 Mbp, 2) chr 5–140.7 Mbp in F2 [Dahl S x Lew] males (8, 11) and BP-f1 at chr 5–146.7 Mbp, and 3) chr 12–28.1 Mbp in F2 [Dahl S x WKY] males (27) and BP-f3 at chr 12–12.8 Mbp (Table 5). With a common hypertensive F2 parental strain and varied normotensive F2 parental strains, reversal of sex specificity strongly suggests the hypothesis that there are genetic modifiers present in normotensive genetic backgrounds. Furthermore, because F2 intercross cohorts exhibiting reversal of sex specificity derive from different normotensive F2 parental strains, cumulative observations suggest that there are multiple genetic modifiers acting on different BP susceptibility QTLs.

Experimental insight into concepts and paradigms.
Detection of F2 intercross-specific QTLs across different studies cumulatively suggests polygenic hypertension subtypes in rat models of hypertension, thus positing a genetic mechanism that could account for clinical heterogeneity in human hypertension and would therefore provide a genotype-based stratification scheme. We note, however, that differences in phenotype characterization may account for differences in QTLs detected. Detection of intercross-common QTL regions, on the other hand, is also important, as they could represent major hypertension susceptibility loci eliciting phenotype regardless of genetic background modifiers, or, alternatively, they could represent QTL clusters (21).

More significantly, however, although susceptibility genes remain to be identified, data show that the conceptual framework of distinct genetic determinants of sex-specific susceptibility to hypertension is unequivocal in the Dahl S polygenic hypertension rat model. These data support the hypothesis that, in addition to modulation of phenotype (30), sex-based microenvironments play a critical role in the determination of hypertension susceptibility genes or the "culprit genotype" per se. These data also raise the question as to whether postmenopausal microenvironments will simply exacerbate premenopausal culprit genotypes or induce a switch in genetic determinants of hypertension susceptibility. Comparative analyses reveal the complexity of sex-specific genetic determinants given that some female-specific QTLs detected in our F2 [Dahl S x R] intercross are sex common or overlap with QTLs detected in other male F2 [Dahl S x non-Dahl R] intercrosses, consistent with the hypothesis of strain-specific genetic modifiers modulating sex-specific susceptibility.

Sex-specific QTLs have been described for diverse traits such as alcohol preference (13), emotionality (36), peak bone mass (34), cholesterol and triglyceride levels (3), and rheumatoid arthritis (1), thus strengthening support for the hypothesis that sex-specific microenvironments alter primary genetic determinants and not just modulate secondary pathogenic events. More importantly, although sex-specific QTLs and interacting loci and strain-specific genetic modifiers of sex-specific determinants underscore the complexity in the elucidation of genetic susceptibility to hypertension and associated target organ complications, their detection implicates multiple genetic mechanism, thus providing confidence in observations and further substantiating the need for a paradigmatic shift toward the independent investigation in males and females of mechanisms, intervention, and prevention strategies for polygenic hypertension and its target organ complications.


   GRANTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by grants from the NHLBI (HL-58136) and Philip Morris External Research Fund. Genotyping was done through the NHLBI Mammalian Genotyping Service at the Center for Medical Genetics, Marshfield Medical Research Foundation (Marshfield, WI).


   FOOTNOTES
 
Address for reprint requests and other correspondence: N. Ruiz-Opazo, Whitaker Cardiovascular Inst., W609, Boston Univ. School of Medicine, 700 Albany St., Boston MA 02118 (e-mail: nruizo{at}bu.edu)

1 The online version of this article contains supplemental data. Back


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 GRANTS
 REFERENCES
 

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