Alan Y. Deng

Genetic mapping of two new blood pressure quantitative trait loci in the rat by genotyping endothelin system genes.

The endothelin system, consisting of a series of potent vasoconstrictor peptides and their receptors, is potentially important in the control of blood pressure. We found that the gene coding for endothelin-2 (ET2), also known as vasoctive intestine peptide, cosegregated strongly with systolic blood pressure in a F2 population [F2(S x LEW)] derived from a cross of the Dahl salt-sensitive (S) rat and the Lewis (LEW/NCrlBR) (LEW) rat. The ET2 locus was assigned to rat chromosome 5. The testis-specific histone (HITH) locus also strongly cosegregated with blood pressure in the F2(S x LEW) population and was assigned to rat chromosome 17. Genetic maps of the regions containing the quantitative trait loci (QTL) for blood pressure on chromosomes 5 and 17 were constructed and the QTL were localized using the MAPMAKER/QTL program. The rat genes for endothelin-1, endothelin-3, and endothelin receptor A did not cosegregate with blood pressure in several F2 populations tested and were assigned to rat chromosomes 17, 3, and 19, respectively. Endothelin receptor B cosegregated weakly with blood pressure and was provisionally assigned to rat chromosome 15. We conclude that, in the rat, one new blood pressure QTL is located on chromosome 5 marked by the ET2 locus and another new QTL is located on chromosome 17 near the HITH locus.

Detection and Positional Cloning of Blood Pressure Quantitative Trait Loci: Is It Possible? Identifying the Genes for Genetic Hypertension

Identification of the quantitative trait loci that influence blood pressure and cause genetic hypertension is a major challenge. Several genetically hypertensive rat strains exist and can be used to locate by linkage analysis broad chromosomal regions containing blood pressure quantitative trait loci. Such broad chromosomal regions, and the narrower subregions, can be moved among strains (ie, production of congenic strains and congenic substrains) to identify small chromosomal regions containing the blood pressure quantitative trait loci. However, ultimate positional cloning of the quantitative trait loci presents a major difficulty because the genetic variants involved are likely to result in subtle changes in function rather than the blatant loss of function characteristic of all mendelian disease genes discovered so far by positional cloning.

Mapping of a quantitative trait locus for blood pressure on rat chromosome 2.

A genetic map for rat chromosome 2 that includes five candidate genes for blood pressure regulation was constructed in a region containing a quantitative trait locus (QTL) for blood pressure. Two F2 populations of male rats raised on high salt (8% NaCI) diet from weaning were studied: F2(WKY x S), derived from a cross of Dahl salt-sensitive rats (S) and Wistar-Kyoto rats (WKY); and F2(MNS x S), derived from a cross of S rats and Milan normotensive strain (MNS). In both populations a blood pressure QTL was localized between Na+,K(+)-ATPase alpha 1 isoform and calmodulin-dependent protein kinase II-delta loci. The LOD score for existence of this blood pressure QTL based on the combined populations (n = 330) was 5.66 and accounted for 9.2% of the total variance and 26% of the genetic variance.

Construction of a double congenic strain to prove an epistatic interaction on blood pressure between rat chromosomes 2 and 10.

Previously we presented suggestive evidence from an F2 segregating population for an interaction on blood pressure (BP) between quantitative trait loci (QTL) on rat chromosomes (Chr) 2 and 10. To prove the existence of such an interaction, we developed congenic strains for Chr 2 and 10 by introgressing the low BP QTL alleles into the Dahl salt-sensitive (S) strain. A double congenic strain was also constructed with both the Chr 2 and 10 low BP QTL alleles on the S background. The four strains (S, Chr 2 congenic, Chr 10 congenic, and Chr 2/10 double congenic) were studied for BP response to increased salt intake. An analysis of variance showed significant main effects of Chr 2, Chr 10, and a significant interaction between Chr 2 and 10 on BP and heart weight (all P < 0.0001). The interaction accounted for 24 mmHg of BP and 79 mg of heart weight. Thus, the discovery and proof of epistatic interactions are clearly critical to understanding the genetics of blood pressure.

Genetic mapping of two blood pressure quantitative trait loci on rat chromosome 1.

A genetic map for rat chromosome 1 was constructed using 66 microsatellite markers typed on either or both of two populations derived from inbred Dahl salt-sensitive (S) rats: F2(LEW x S) n = 151, and F2(WKY x S) n = 159. These populations had been raised on a high salt (8% NaCl) diet. Systolic blood pressure and heart weight were found to be genetically linked to two separate regions on rat chromosome 1 in the F2(LEW x S) population. One region was centered around the anonymous SA locus and accounted for 24 mmHg of blood pressure. The other region was 55 cM from the SA locus centered around a cluster of cytochromes P450 loci, and accounted for 30 mmHg of blood pressure. Since blood pressure and heart weight were highly correlated these same regions were also linked to heart weight. These results were cross-specific as linkage of these chromosome 1 regions to blood pressure and heart weight was not observed in several other F2 populations derived by crossing S and other normotensive control strains. This is presumably due to different alleles and/or different genetic backgrounds in the various populations. The SA region of chromosome 1 was found to influence body weight in F2(LEW x S) rats. Combining the present data with our previously published data on the F2(LEW x S) population showed that four separate quantitative trait loci with additive effects accounted for 106 mmHg and 38% of the total variance of blood pressure and for 506 mg and 34% of the total variance of heart wt.

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.

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

Chromosomal assignment of 11 loci in the rat by mouse-rat somatic hybrids and linkage

Eleven rat genes have been assigned to rat chromosomes by use of mouse × rat somatic hybrids and/or use of linkage to known chromosome markers. Among them, the genes for the inducible nitric oxide synthase (Nos2) and for a vasoactive intestinal peptide receptor (Vipr) are potential candidates for genetic regulation of blood pressure and were localized to rat Chromosomes (Chrs) 10 and 8 respectively. Genes for gastric H,K-ATPase alpha subunit (Atp4a). Class I alcohol dehydrogenase (Adh), and aldolase C (Aldoc) were localized to Chrs 1, 2, and 10 respectively, and thus provide more DNA markers for genetic mapping of quantitative trait loci for blood pressure on those chromosomes. Genes for alkaline phosphatase (Alp1) and cardiac AE-3 Cl-/HCO3- exchanger (Ae3) were both localized to Chr 9. Genes for glutamate dehydrogenase (Glud) and gastric H,K-ATPase beta subunit (Atp4b) were localized to Chr 16. The ornithine decarboxylase (Odc) gene and ornithine decarboxylase pseudogene (Odcp) were localized to Chrs 6 and 11 respectively.