Howard Dene

Linkage map and congenic strains to localize blood pressure QTL on rat Chromosome 10

Our purposes were to develop a linkage map for rat Chromosome (Chr) 10, using chromosome-sorted DNA, and to construct congenic strains to localize blood pressure quantitative trait loci (QTL) on Chr 10 with the map. The linkage mapping panel consisted of three F2 populations totaling 418 rats. Thirty-two new and 29 known microsatellite markers were placed on the map, which spanned 88.9 centiMorgans (cM). The average distance between markers was 1.46 cM. No markers were separated by more than 6.8 cM. Four congenic strains were constructed by introgressing various segments of Chr 10 from the Milan normo-tensive strain (MNS) onto the background of the Dahl salt-sepsitive (S) strain. A blood pressure QTL with a strong effect on blood pressure (35-42 mm Hg) when expressed on the S background was localized to a 31-cM region between DIOMco6 and DIOMcol. The region does not include the locus for inducible nitric oxide synthase (Nos2), which had been considered to be a candidate locus for the QTL.

Time-Course Genetic Analysis of Albuminuria in Dahl Salt-Sensitive Rats on Low-Salt Diet

The Dahl salt-sensitive hypertensive (S) rat develops albuminuria early in life even on a low-salt diet. In contrast, the spontaneously hypertensive rat (SHR) is highly resistant to developing albuminuria despite elevated BP. An F(1) hybrid of S and SHR showed a low urinary albumin excretion (UAE) and low urinary protein excretion (UPE) similar to SHR, i.e., SHR was dominant. A genetic analysis was carried out on a large population (n = 276) obtained by backcrossing F(1) rats to the recessive S strain; the population was fed a low-salt diet. Genome scans done at 8, 12, and 16 wk of age yielded ten quantitative trait loci (QTL) for UAE and/or UPE with variable time-course patterns on nine rat chromosomes (RNO), i.e., RNO1, RNO2, RNO6, RNO8, RNO9, RNO10, RNO11, RNO13, and RNO19. There were two UPE QTL on RNO6. At most of the UAE and/or UPE QTL, the S allele was associated with increased excretion, except for one of the QTL on RNO6 and the QTL on RNO11, where the S allele caused decreased excretion. Only the UAE and UPE QTL on RNO10 co-localized with a BP QTL. The S allele on RNO10 caused higher BP and higher UAE. Two additional BP QTL were detected on RNO1 and RNO6. Most of the UAE and UPE QTL co-localized with QTL for kidney lesions characteristic of S rats. Multiple interactions were observed for UAE, many of which involved RNO2. In summary, UAE is highly polygenic and the majority of the QTL altering UAE do not co-localize with QTL for BP as evaluated by tail-cuff measurements of BP.

Congenic Strains for the Blood Pressure Quantitative Trait Locus on Rat Chromosome 2

A quantitative trait locus (QTL) for blood pressure was previously detected by linkage analysis in a region of rat chromosome 2 using segregating populations derived from crosses of the Dahl salt-sensitive (Dahl S) rat with rats of the Wistar-Kyoto (WKY) strain or Milan normotensive strain (MNS). Two congenic strains, S.WKY-D2N35/Nep and S.MNS-Adh/D2Mit5, have been constructed by replacing a region of chromosome 2 of the Dahl S rat with the homologous region (ie, low blood pressure QTL allele) of either the WKY or MNS rat, respectively. Systolic pressures of congenic strains S.WKY-D2N35/Nep and S.MNS-Adh/D2Mit5 fed a 2% NaCl diet for 24 days were 44+/-4.6 and 29+/-4.5 mm Hg lower, respectively, than that of the comparably treated Dahl S rats. The differences between congenic and Dahl S rats in blood pressure were highly significant (P<.001) and were corroborated by significantly (P<.001) lower ratio of heart weight to body weight in the congenic strains compared with Dahl S rats. The data from two congenic strains combined unequivocally establish the existence of a blood pressure QTL on rat chromosome 2.

Interval mapping and congenic strains for a blood pressure QTL on rat Chromosome 13

The rerun locus (Ren) on rat Chromosome (Chr) 13 had previously been shown to cosegregate with blood pressure in crosses involving Dahl salt-sensitive (S) and Dahl salt-resistant (R) rats. In the present work, interval mapping of blood pressure on Chr 13 with a large F2 (S x R), n = 233, population yielded a maximum LOD = 4.2 for linkage to blood pressure, but the quantitative trait locus (QTL) was only poorly localized to a large 35-centiMorgan (cM) segment of Chr 13. In the linkage analysis, the S-rat QTL allele (S) was associated with higher, and the R-rat QTL allele (R) with lower blood pressure, the difference between homozygotes being about 20 mm Hg. A congenic strain was made by introgressing the R-rat Ren allele into the recipient S strain. This congenic strain showed a 24 mm Hg reduction (P = 0.004) in blood pressure compared with S rats for rats fed 2% NaCl diet for 24 days; this difference was confirmed by two other independent tests. Two congenic substrains were derived from the first congenic strain with shorter R Chr 13 segments on the S background. Comparisons among these congenic strains showed that a blood pressure QTL was in the 24-cM chromosomal segment between Syt2 and D13M1Mit108. This segment does not include the renin locus, which is thus excluded from being the gene on rat Chr 13 responsible for genetic differences in blood pressure detected by linkage analysis.

Seven renin alleles in rats and their effects on blood pressure.

Objective To survey inbred rat strains for renin alleles and to test those alleles for effects on blood pressure Design Rat strains with renin alleles different from the s allele carried by inbred Dahl salt-sensitive (SS/Jr) rats were crossed with SS/Jr rats and subsequently intercrossed to produce F2 populations. Thus, in each F2 population segregation of the renin allele and a contrasting renin allele occurred. F2 rats were raised on a high-salt diet and their blood pressures were determined. The cosegregation of renin alleles with blood pressure was evaluated in each F2 population Methods Renin alleles were recognized by the variable number of tandem repeats in the first intron of the renin gene and by a variable Hind III site in the fifth intron. Genotyping was by restriction fragment length polymorphism analysis Results Seven renin alleles were found, but breeding stock for only six of these alleles was available. From these stocks it was possible to construct five F2 populations in which the s renin allele segregated with a contrasting renin allele. In all five F2 populations the rats homozygous for the s allele had higher blood pressure than those homozygous for the contrasting renin allele, but statistical significance was most easily established in the F2 intercrossed offspring of an SS/Jrx inbred Dahl salt-resistant (SR/Jr) rat cross. This result was duplicated Conclusions There are at least seven alleles in rats at the renin locus, allowing many pairwise allelic comparisons to be made. Of the five alleles compared with the s renin allele, the r allele of SR/Jr rats was unique in providing strong evidence for cosegregation with blood pressure. The naive expectation that all crosses should yield the same cosegregation result with blood pressure for a candidate locus is not consistent with either theory or the present experimental results

Blood pressure and survival of a Chromosome 7 congenic strain bred from Dahl rats

Abstract11 β-hydroxylase (Cyp11b1) mutations were previously linked to altered steroid biosynthesis and blood pressure in Dahl salt-resistant (R) and Dahl salt-sensitive (S) rats. In the present work, interval mapping identified a putative blood pressure quantitative trait locus (QTL) near Cyp11b1 in an F1(SxR)xS population (LOD = 2.0). Congenic rats (designated S.R-Cyp11b) were constructed by introgressing the R-rat Cyp11b1 allele into the S strain. S.R-Cyp11b rats had significantly lower blood pressure and heart weight compared with S rats, proving the existence of a blood pressure QTL on Chromosome (Chr) 7 despite the fact that QTL linkage analysis of blood pressure never achieved stringent statistical criteria for significance. To test the effects of the introgressed region on blood pressure and survival, S.R.-Cyp11b and S rats were maintained on a 4% NaC1 diet until they died or became moribund. Analysis of variance (ANOVA) indicated significant strain differences in blood pressure and days survived (P < 0.0001 for both) as well as gender differences in days survived (P = 0.0003). Kaplan-Meier survival analysis also found significant strain (P < 0.0001) and gender (P = 0.007) differences in days survived. However, when the effects of blood pressure were removed, significant strain differences in survival essentially disappeared. This suggests that the increased survival of S.R-Cyp11b rats was largely due to their decreased blood pressure and thus strongly corroborates the existence of a blood pressure QTL on Chr 7 near or at Cyp11b1.

Localization of a blood pressure QTL to a 2.4-cM interval on rat chromosome 9 using congenic strains.

A blood pressure (BP) quantitative trait locus (QTL) was previously found on rat chromosome 9 using Dahl salt-sensitive (S) and Dahl salt-resistant (R) rats. A congenic strain, S.R(chr9), constructed by introgressing an R chromosomal segment into the S background, previously proved the existence of a BP QTL in a large 34.2-cM segment of chromosome 9. In the current work congenic substrains were constructed from the progenitor congenic strain, S.R(chr9). BP and heart weight comparisons between these congenic substrains and their S control localized the BP QTL to a 4.6-cM interval. Two solute carrier (Na(+)/H(+) exchanger) genes, Nhe2 and Nhe4, were excluded as candidates based on their map locations. A second iteration of congenic substrains was used to localize the QTL further to a 2.4-cM interval. Another solute carrier (Cl(-)/HCO3- exchanger) gene, Ae3, is in this reduced interval and was sequenced for both S and R strains, but no coding sequence variations were found. Ae3 mRNA was not differentially expressed in the kidney of congenic compared to S rats. Although the identity of the QTL remains unknown its map location has been reduced from an interval of 34.2 to 2.4 cM.

Lack of Effects of Maternal Salt Intake on Blood Pressure of Offspring in Dahl Salt-Sensitive Rats

Inbred Dahl salt sensitive (S/JR) and salt resistant (R/JR) rats were used to look for effects of varying maternal intake of salt (NaCl) on the blood pressure of the offspring. Neither the blood pressure at weaning nor the blood pressure response to postweaning high salt diet of S/JR or R/JR rat pups was affected whether their mothers had eaten high salt (8% NaCl) or low salt (0.15% NaCl) diet during gestation. Similarly, maternal salt intake during lactation had no effect on the blood pressure of the offspring at weaning or the blood pressure response to salt feeding after weaning. Na+ was higher and K+ was lower in milk of S/JR compared to R/JR mothers during the last half of the lactation period, but dietary salt intake did not influence milk Na+ or K+. Previous cross-fostering experiments show that this strain difference in milk electrolytes does not influence S pups blood pressure. It is concluded that neither maternal salt intake nor the existing changes in milk Na+ have any influence on the blood pressure of Dahl salt sensitive rat pups in contrast to the marked effects of salt intake in these rats after weaning.

Genetic analysis of alpha2-adrenergic receptors and blood pressure using Dahl salt-sensitive rats

Objective To evaluate the role of α2-adrenergic receptors in genetic hypertension by cosegregation analysis using Dahl rats Design Inbred Dahl salt-sensitive (SS/Jr) rats were crossed with inbred Dahl salt-resistant (SR/Jr) rats; also, SS/Jr rats were crossed with several control strains, and large F2 populations were subsequently produced from each cross. All F2 populations were raised on a high-salt diet. The rats were genotyped, where possible, at the loci for three different subtypes of α2-adrenergic receptors designated as classes I, II and III. The blood pressures of the rats classified by genotype at each α2-adrenergic receptor subtype locus were compared using analysis of variance Methods Genomic clones of three classes of α2-adrenergic receptors were isolated from genomic γ-phage libraries of SS/Jr or SR/Jr rat strains, or both, by screening with complementary DNA for human α2-adrenergic receptors. Fragments of the rat genomic clones obtained were used for genotyping by restriction fragment length polymorphism. Also, cloned genomic DNA flanking the α2-adrenergic receptors and containing microsatellites was sequenced; genotyping at informative microsatellite markers was performed using the polymerase chain reaction. Two of the three classes of rat α2-adrenergic receptors were localized to rat chromosomes by linkage analysis or using a panel of mouse-rat hybrid somatic cell lines Results Rat α2-adrenergic receptor classes I and III genes were assigned to rat chromosomes 14 and 3, respectively. These correspond to α2-adrenergic receptor genes on human chromosomes 4 and 2, respectively. Extensive cosegregation analysis, involving five alleles in six segregating populations for class I α2-adrenergic receptors, yielded no evidence of an effect of these loci on blood pressure. Classes II and III α2-adrenergic receptors could each be tested in only one population and there was no evidence for an effect of either receptor gene on genetic differences in blood pressure. The dopamine-IB receptor was closely linked to the class I α2-adrenergic receptor on rat chromosome 14. Thus, the negative cosegregation of the class I receptor with blood pressure applies equally to the dopamine-1 B receptor Conclusions Genetic analysis in segregating populations involving crosses of inbred Dahl salt-sensitive rats with five other strains provides no evidence for a genetic effect of class I α2-adrenergic receptors, or of the dopamine-1 B receptor, on blood pressure. Classes II and III α2-adrenergic receptors also failed to cosegregate with blood pressure but, because only limited testing was possible with the classes II and III receptors, this negative result is not definitive

Restriction fragment length polymorphisms for the renin gene in Dahl rats.

Genomic DNAs from inbred Dahl salt-hypertension sensitive (S) and inbred Dahl salt-hypertension resistant (R) rats were examined for restriction fragment length polymorphisms (RFLPs) using a rat renin cDNA probe. Eight of the 28 restriction enzymes used yielded polymorphic fragments between S and R strains. This suggests a major structural difference in or near the renin gene of Dahl rats. Some, but not all, of the polymorphisms are compatible with an insertion/deletion mutation of about 1.1 kb in size. The ratio of renin gene copy numbers for S to R was found to be 1, showing that the RFLPs are not likely to be due to gene duplication.