Role of Chromosome X in the Sabra Rat Model of Salt-Sensitive Hypertension
Abstract—We carried out a total genome screen in the Sabra rat model of hypertension to detect salt-susceptibility genes. We previously reported in male animals the presence of 2 major quantitative trait loci (QTLs) on chromosome 1 that together accounted for most of the difference in the blood pressure (BP) response to salt loading between Sabra hypertension-prone rats (SBH/y) and Sabra hypertension-resistant rats (SBN/y). In females, we reported on 2 major QTLs on chromosomes 1 and 17 that together accounted for only two thirds of the difference in the BP response between the strains. On the basis of phenotypic patterns of inheritance in reciprocal F2 crosses, we proposed a role of the X chromosome. We therefore continued the search for the missing QTL in females that would account for the remaining difference in the BP response between the 2 strains using newly developed microsatellite markers and focusing on chromosome X. We screened an F2 cross, consisting of 371 females and 336 males, using 19 polymorphic chromosome X microsatellite markers. We analyzed the averages of BP by genotype using ANOVA and the individual data using MAPMAKER/QTL. In female F2 progeny, we identified a segment on chromosome X that spans over 33.4 cM and shows significant cosegregation (P<0.001) of 14 microsatellite markers (demarcated by DXRat4 and DXMgh10) with systolic BP after salt loading. This segment has 2 apparent peaks at DXRat4 and DXRat13, with a BP effect of 14 mm Hg for each. Multipoint linkage analysis with a free model detected 3 peaks (logarithm of the odds ratio [LOD] score >4.3) within the same chromosomal segment: One between DXMgh9 and DXMit4 (LOD 4.9; 6.1% of variance), a second between DXMgh12 and DXRat8 (LOD 5.2; 7.2% of variance), and a third between DXRat2 and DXRat4 (LOD 5.8; 7.5% of variance). On the basis of these findings and until congenic strains become available, our working assumption is that within chromosome X, 1 to 3 genetic loci contribute importantly to the BP response of female Sabra rats to salt. In male F2 progeny, we detected no significant cosegregation of any region on chromosome X with the BP response to salt loading. We conclude that in the female rat, salt susceptibility is mediated by 3 to 5 gene loci on chromosomes 1, 17, and X, whereas in the male rat, the X chromosome does not affect the BP response to salt.
We previously carried out a total genome scan in the Sabra rat model of hypertension to detect salt-susceptibility genes.1 We reported in males the presence of 2 quantitative trait loci (QTLs) on chromosome 1 and in females of one QTL on chromosome 1 (QTL1b) and a second QTL on chromosome 17 (QTL17). Using an additive model of gene effect on phenotype, we calculated that the QTL found in males could account for all of the genetic difference in the blood pressure (BP) response to salt loading between Sabra hypertension-prone rats (SBH/y) and Sabra hypertension-resistant rats (SBN/y). In females, the additive effect of the 2 QTLs accounted for not more than two thirds of the difference in the BP response to salt loading between SBN/y and SBH/y. On the basis of these findings, we concluded that in females we had not detected all of the salt-susceptibility gene loci. We also observed and reported in the crosses between SBH/y and SBN/y an unusual mode of inheritance of salt susceptibility that was also highly suggestive of an involvement of chromosome X.2
In the current study, we continued to search in the female rat for the missing QTLs that would account for the remaining difference in the BP response to salt loading between SBH/y and SBN/y using newly developed microsatellite markers and focusing on chromosome X. Specifically, we scanned the X chromosome of the Sabra rat for genes that cosegregate with salt sensitivity and/or salt resistance in terms of the development of hypertension.
SBH/y and SBN/y (Barzilai Medical Center colony, Ashkelon, Israel)3 were crossbred. The initial cross (cross 1a) was between 7 female SBH/y and 2 male SBN/y. To ensure reproducibility, this cross was repeated (cross 1b) between 3 female SBH/y and 1 male SBN/y. The reciprocal cross (cross 2a) was between 3 female SBN/y and 1 male SBN/y and was repeated (cross 2b) between 5 female SBN/y and 2 male SBN/y. The F1 progeny from each cross were inbred (brother-sister mating) to produce F2 generations. The F2 populations were weaned at 1 month of age.
The animals were housed in the center’s animal facility in strict compliance with institutional regulations and following the guidelines set forth by the American Physiological Society. Climate-controlled conditions were maintained, and temperature was set at 22°C. Regular 12-hour diurnal cycles were kept using an automated light/dark switching device. Unless stated otherwise, tap water and standard rat chow containing 0.65% NaCl (Koffolk) were provided ad libitum.
At 6 weeks of age, basal BP (with rats on standard chow and before salt loading) was measured. Animals were then salt loaded by implanting subcutaneously in the back of the neck a 25-mg deoxycorticosterone acetate pellet (Innovative Research) and providing 1% NaCl as drinking water (hereforth, “salt loading”). Animals were maintained on standard rodent chow ad libitum. After 4 weeks of salt loading, BP was measured for the second time.
Systolic BP was measured at ambient temperature (27° to 28°C) in awake animals by the tail-cuff method using a photoelectric oscillatory detection device (IITC Life Sciences), as previously described.3 At least 3 replicate BP measurements were made by the same operator on 3 consecutive days (ie, at least 9 measurements over 3 days), both at baseline and after salt loading. The average of all measurements at each experimental time point was taken as representative of systolic BP.
Genomic DNA was prepared from tail clippings of each animal by salt precipitation, followed by phenol-chloroform cleaning, as previously described.3 Purity and quantity of extracted DNA were assessed spectrophotometrically (GeneQuant II, Pharmacia Biotech).
Microsatellite markers for chromosome X (http://ratmap.gen.gu.se) were obtained from Research Genetics. Of the 34 rat chromosome X markers tested, 4 did not amplify, 11 were not polymorphic between the strains and thus not informative, and 19 produced polymorphic bands when DNA samples from SBH/y and SBN/y were amplified by polymerase chain reaction (PCR).
Genotyping was carried out by PCR amplification, as previously described.3 In brief, genomic DNA (50 ng) was amplified by PCR. The forward primer was labeled with [32P]ATP (Dupont NEN) using T4 polynucleotide kinase (Promega). The PCR reactions were processed on a PTC 100 thermal cycler (MJ Research). The product of each reaction (3 μL) was loaded onto a 7% polyacrylamide gel and run using a Base Ace apparatus (Stratagene) at 60 W (Feathervolt 3000, Stratagene) for 4 hours and exposed to Kodak XAR-5 film for autoradiography.
Linkage and Statistical Analysis
Data were analyzed initially to determine whether basal BP and BP after salt loading in the F2 progeny cosegregated with the chromosome X markers tested. For the purposes of analysis, BP data from crosses 1a and 1b and crosses 2a and 2b were combined. Analysis was carried out in separate in females and males for the effect of genotype on phenotype (systolic BP) by one-way ANOVA (Complete Statistical Software, StatSoft). Summary data are represented as mean±SEM. When cosegregation was found, multipoint linkage analyses were carried out using the MAPMAKER/EXP 3.0 and MAPMAKER/QTL 1.1 programs.4 5 6 Since genotype distributions at all of the X chromosome molecular markers tested were approximately at a ratio of 1:1:2 in females for the homozygous (HH and NN) and heterozygous (HN) states, respectively, the data were fed into the program as an F2 intercross.
The F2 cohort resulting from the crosses between SBH/y and SBN/y consisted of a total of 707 animals: 371 female and 336 male. In cross 1, in which female SBH/y were crossed with male SBN/y, the resulting F2 progeny consisted of 178 male and 211 female rats. In cross 2, in which male SBH/y were crossed with female SBN/y, the resulting F2 progeny consisted of 158 male and 160 female rats.
The linkage map for the chromosome X data is shown in Figure 1⇓. The map was constructed using 18 markers, covering 49.8 cM, which is 40.8% of the estimated size of chromosome X (http://ratmap.gen.gu.se/chromap.html). The logarithm of the odds ratio (LOD) tracings for chromosome X for females is shown in Figure 2⇓.
Analysis of basal BP by genotype for female and male F2 cohorts at each of the tested chromosome X marker loci revealed no cosegregation by ANOVA nor linkage by analysis with MAPMAKER/QTL (data not shown).
BP Response to Salt Loading
In the female cohort, analysis by ANOVA revealed on chromosome X significant cosegregation (P<0.001) of 14 microsatellite markers (demarcated by DXRat4 and DXMgh10) with BP after salt loading. There were 2 apparent peaks, one near the telocentromere at DXRat4 and the other at DXRat13, each with a similar BP effect of 14 mm Hg (Table 1⇑). Multipoint linkage analysis of the female F2 cohort using the MAPMAKER/QTL programs detected 3 peaks (LOD>4.3) (Figure 2⇑) between DXRat4 and DXMgh10: One between DXMgh9 and DXMit4 (LOD 4.946; 6.1% of variance), a second between DXMgh12 and DXRat8 (LOD 5.137; 7.2% of variance), and a third between DXRat2 and DXRat4 (LOD 5.823; 7.5% of variance). In the male F2 progeny, ANOVA (Table 2⇑) revealed no significant cosegregation with the BP response to salt loading of any region on chromosome X.
Mode of Transmission of Salt Susceptibility
Analyses of the pedigrees leading from the parent progenies through the F1 to the F2 generations revealed a highly unusual and complex mode of transmission. In cross 1 (female SBH/y with male SBN/y), the BP response to salt loading of the F2 cohort (n=389) revealed a bimodal BP distribution that was explained in part by differences in BP distribution between males (n=178) and females (n=211). About two thirds of the males tended to have a BP response in the higher range and one third in the lower range, whereas two thirds of the females had a BP response in the lower range and one third in the higher range (data not shown). In cross 2, the reciprocal cross (male SBH/y with female SBN/y), the BP response to salt loading in the F2 cohort (n=318) was bimodal in males (n=158), with about half of the animals having a lower response and half having a hypertensive response; in females (n=160), nearly all BP measurements were in the low range (data not shown).
Our present data were collected as part of a continuing effort to scan the rat genome for salt-susceptibility genes. On the basis of our previous findings,1 2 we targeted the X chromosome at this time. The study was conducted in a large F2 cohort bred from a cross between SBH/y and SBN/y. We detected cosegregation of a 33.4-cM segment of chromosome X, extending from the telocentromere region and spanning to and through the q2 band of the chromosome. On the basis of the current ANOVA and linkage analyses, it is not possible to determine whether this region contains 1, 2, or 3 separate QTLs. Until congenic and subcongenic strains become available, the working assumption is therefore that there are, within the segment of the X chromosome defined in this study, 1 to 3 genes that contribute to the genetic variance of the BP response to dietary salt intake in females but not in males. Thus, salt susceptibility in the female Sabra rat maps, in addition to chromosomes 1 and 17, to one or more genetic loci on chromosome X.
In the course of the study, we constructed a genetic map of chromosome X that spans over 49.8 cM, using 18 informative microsatellite markers. This genetic span is close to that reported by Jacob et al7 and less than that reported by Millwood et al,8 who published a map of chromosome X that spans over a genetic distance of 88.1 cM. This latter map was derived from integrated data from 5 intercrosses and by using mostly Wox microsatellite markers. If we overlap the map generated from the current study onto the Millwood map,8 the difference in the calculated map size narrows down to 13.1 cM. Thus, our current map of chromosome X is fairly consistent with other published maps.7 8 In either case, considering the estimated full size of chromosome X at 122 cM (http://ratmap.gen.gu.se/chromap.html), none of the currently available maps cover the entire chromosome.
Comparison of the current findings with respect to salt susceptibility–related gene loci in other crosses and strains reveals only one parallel in which a linkage study detected a BP QTL on chromosome X. Hilbert et al9 studied an F2 cross between stroke-prone spontaneously hypertensive and normotensive Wistar-Kyoto rats. Their results with respect to the X chromosome were complex and consisted of one locus accounting for part of the variability of the basal systolic BP in female animals, of no segregation with BP after salt loading in females, and of partial segregation in males depending on the Y chromosomal parental status. In Dahl rats, the other major genetic model of salt susceptibility, no QTL has been reported so far on the X chromosome.
Seventeen genes can be identified in the areas swept by the placement confidence intervals of the linked microsatellite markers on chromosome X in the female Sabra rat (http://ratmap.gen.ge.se/lassolite/Lasso.acgi). On the basis of current knowledge of pathophysiological mechanisms of salt susceptibility, none of these stand out at this time as obvious candidate genes for salt susceptibility. Of note, though, are the angiotensin II receptor and the vasopressin receptor V2 genes, 2 highly attractive genes candidate genes that are located on the X chromosome but outside the chromosome segment that was found to cosegregate with the BP response to salt loading in our crosses.
The present data confirm and reinforce the existence of sexual dimorphism in the genetic basis of salt susceptibility that we previously reported in the Sabra model of salt susceptibility with respect to the QTLs on chromosomes 1 and 17.1 The current findings indicate that the QTLs on the X chromosome are sex-specific for females. Since in the female Sabra rat, chromosomes 1 and 17 contribute together to nearly 70% of the BP response to salt loading, calculations based on an additive model of quantitative genetic analysis suggest that the additional 14 mm Hg contributed by this chromosome (assuming at least one QTL) account for an additional 26% of the difference in the BP response to salt loading between SBH/y and SBN/y. Thus, the combined effects of the QTLs SS-1b, SS-17, and those on chromosome X account for most of the overall phenotype variance in female Sabra rats, leaving only 6% unaccounted for (Figure 3⇓). If, however, more than one culprit gene locus are eventually shown to be present on chromosome X, then all of the phenotypic variance in the female Sabra rat might already be accounted for.
Of particular interest with respect to the role of chromosome X in salt susceptibility is the highly unusual and complex mode of transmission of the BP response to salt loading in the F2 generation of the crosses.2 The lack of a hypertensive response to salt loading in females of cross 2 stands out in the phenotype analysis (Figure 4⇓). Although difficult to interpret, a possible explanation is that in female rats a major salt-resistance dominant allele on the X chromosome modulates expression (inhibitory effect) of the salt-sensitive autosomal genes. The salt-resistance allele on the X chromosome (Xn) appears to be transmitted in a pattern of mendelian dominance, and in its presence, there is a general lack of hypertensive response to salt loading. In the F2 generation of cross 1, females could develop hypertension as only some but not all carry the resistance allele, thus allowing the other salt-susceptibility autosomes to come into action; whereas in cross 2, all females carry the N allele, and thus BP did not rise in response to salt. The exact location of the salt-resistance gene on the X chromosome could not be determined by the methodologies used in the current study, although its presence on the X chromosome is highly suggested.
Finally, the difference in BP observed in the F2 progeny between male and female rats, irrespective of genotype (see Tables 1⇑ and 2⇑), needs to be commented on. In the parental strains (SBH/y and SBN/y), there are no sex differences in basal BP or BP after salt loading within each strain.3 In F2 generations of crosses 1 and 2 in the current study, average BP after salt loading was lower in females than in males. Of note is that BP before salt loading did not differ between the sexes (data not shown). In previous analyses of chromosomes 1 and 17, we made a similar observation.1 At this time, we find this observation of interest but have no clear explanation for the effect of sex on the BP response to salt loading.
In summary, we have detected in a cross between SBH/y and SBN/y a segment on chromosome X that incorporates 1 to 3 QTLs for salt susceptibility. The QTLs are specific for female animals only. Additional work with congenic and subcongenic strains is required to determine whether one or more gene loci are involved. Together, the gene loci detected so far in the female Sabra rat on chromosomes 1, 17, and X appear to account for most, and possibly all, of the BP variance between SBH/y and SBN/y and therefore contribute importantly to the development of hypertension in response to dietary salt exposure. Further exploration of the Sabra model of salt susceptibility, the eventual identification of causative genes, and the decoding of the mechanism of action may ultimately provide clinically relevant tools for novel diagnostic and therapeutic approaches that will benefit the important group of salt-susceptible individuals with hypertension.
This study was supported by grants from the German-Israeli Binational Science Foundation (GIF), the Israel Science Foundation, and Cilag International and by the EUROHYPGEN II Concerted Action of the Biomedical Program of the European Community. The authors acknowledge the contributions of Gurion Katni, Marina Grinyok, and Heidelinde Müller for technical support. We are indebted to Howard Jacob for his advice and continuous support.
- Received September 15, 1998.
- Revision received October 20, 1998.
- Accepted October 29, 1998.
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