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

Methods

Results

Two congenic strains were produced: (1) S.WKY-D2N35/Nep rats were produced by substituting the chromosome 2 region from D2N35 to Nep of the Dahl S rat with the homologous region of the WKY; (2) S.MNS-Adh/D2Mit5 rats were produced by replacing the chromosome region Adh to D2Mit5 of the Dahl S rat with the homologous region of the MNS rat (Figure⇓). The entire region substituted in the S.WKY-D2N35/Nep strain is homozygous for the WKY allele (ie, WW) for all the markers located within the region (Figure⇓, left). The entire region substituted in the S.MNS-Adh/D2Mit5 strain is homozygous for the MNS allele (ie, MM) for all the markers located within the region (Figure⇓, right).

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Figure 1.

Chromosome 2 congenic strains. Maps were derived from genotyping of chromosome 2 markers in an F₂ WKY×S population (left) and F₂ MNS×S population (right) using markers given in references.^{2 10 11} Numbers to the left of the map indicate distances between markers in centimorgans (cM) using the Haldane correction. The order of markers was determined both by linkage and by scoring crossover recombinants during the construction of congenic strains. Solid bars to the right of each map represent the chromosome segment of the Dahl S rat being replaced by the homologous segment of the WKY (left) and MNS (right) rat in the construction of two congenic strains. The entire region indicated by solid bars and junctions between solid and open bars are homozygous for the WKY strain allele (WW, left) and MNS strain allele (MM, right) for all the markers listed in the corresponding positions within the solid bars on the map. Open bars at the ends of solid bars indicate the chromosome regions where crossover break points are between markers in the congenic strains. S.WKY-D2N35/Nep indicates congenic S.WKY-D2N35/neutral endopeptidase strain; S.MNS-Adh/D2Mit5, congenic S.MNS-alcohol dehydrogenase/D2Mit5 strain; Camk, calmodulin-dependent protein kinase II-delta; Nak, Na⁺,K⁺-ATPase α₁; Gca, guanylyl cyclase A/atrial natriuretic peptide receptor A; Fgg, fibrinogen gamma; Nep, neutral endopeptidase; Adh, alcohol dehydrogenase; At1b, angiotensin receptor type 1B; and Prlr, prolactin receptor. D2N35 is an anonymous marker¹⁵ ; D2Mit5, D2Mit8, D2Mit15, and D2Mgh12 are anonymous markers¹⁶ and were purchased from Research Genetics. S indicates the Dahl S strain allele; M, the MNS strain allele; and W, the WKY strain allele.

If there is truly a BP QTL present in the chromosome 2 regions in question, the BP of the rats for both congenic strains should be significantly lower than that of Dahl S rats.^{1 2} The Table⇓ shows pairwise strain comparisons of the congenic rats with Dahl S rats. BPs of congenic S.WKY-D2N35/Nep rats and congenic S.MNS-Adh/D2Mit5 rats were 44±4.6 and 29± 4.5 mm Hg lower than that of Dahl S rats, respectively. These differences are highly statistically significant (P<.001). The ratios of heart weight to body weight of congenic rats were also highly significantly lower than those of Dahl S rats (Table⇓).

In our other linkage studies, an additional BP QTL was also detected in F₂ S×WKY and F₂ S×MNS populations. This QTL is located in a region on rat chromosome 10.^{1 12} To verify that this region on chromosome 10 does not contain any residual genome from WKY or MNS in the two congenic strains developed for chromosome 2, chromosome markers located in the chromosome 10 region of interest were genotyped for the rats of the S.WKY-D2N35/Nep and S.MNS-Adh/D2Mit5 congenic strains. The results showed that the entire chromosome 10 region in question in each congenic strain is homozygous SS. The chromosome 10 QTL is the only other QTL we know about in crosses of Dahl S with MNS or WKY and is the only other region tested.

Discussion

The present data establish a major BP QTL in the rat chromosome 2 region between markers D2N35 and Nep. This is the region shared by both congenic S.WKY-D2N35/Nep and S.MNS-Adh/D2Mit5 strains (Figure⇑). After eight backcrosses used in the production of the congenic strains, the genetic background of the congenic strain outside of the selected region is expected on average to be 99.6% Dahl S genes and 0.4% residual (unwanted) genomic regions of the normotensive donor strain (WKY or MNS). Because the two congenic strains were derived independently, it is very unlikely after eight backcrosses that both strains would by chance contain genomic regions of residual donor-strain genetic material in their genetic backgrounds that would affect BP. The chromosome 10 region, which was implicated as containing a BP QTL in our other linkage studies involving WKY and MNS strains,^{1 12} was shown to be homozygous for the Dahl S rat allele in both congenic strains. Therefore, the BP effect observed for the S.WKY-D2N35/Nep and S.MNS-Adh/D2Mit5 congenic strains (Table⇑) can be attributed to the chromosome 2 region between the D2N35 and Nep markers.

BP differences between congenic and Dahl S rats were corroborated by differences in heart weight–body weight ratios. It is noteworthy that the only valid comparisons are within experiments, not between experiments. Thus although, for example, BP was consistent in the Dahl S strain between experiments (205.8±3.91 versus 209.8±4.11 mm Hg), heart weight–body weight ratio (3.80±0.047 versus 4.23±0.054) was not (Table⇑). The latter is attributed to different people dissecting the hearts between experiments. In our studies, we use heart weight–body weight ratio as an index for corroborating chronic BP differences. In all our linkage and congenic studies so far carried out, heart weight and BP correlate very well (Reference 17¹⁷ ; Table⇑, current study; and unpublished data, 1996). It is also likely, however, that heart weight is influenced by loci that act independently of BP QTL.^{18 19 20 21}

Although there are differences in the precise protocol used for genotyping, BP effects of congenic strains in the present study are consistent with those of cosegregation studies using F₂ populations. In both F₂ WKY×S and F₂ MNS×S populations, the rats homozygous for the WKY or MNS QTL allele had BP lower than the rats homozygous for the Dahl S QTL allele.² Likewise, the BPs of both congenic strains were also lower than that of Dahl S rats (Table⇑).

Theoretically, there could be more than one BP QTL located in the chromosome 2 region in question. Consequently, the BP effect observed in each congenic strain could come from the effects of these separate QTL. This possibility of having multiple QTL in the congenic region will be resolved by constructing congenic substrains, each one containing a smaller substituted segment within the broad chromosome 2 region under study.

The BP reduction of the congenic S.WKY-D2N35/Nep strain is about 15 mm Hg more than that of the congenic S.MNS-Adh/D2Mit5 strain (Table⇑). This difference could arise if the QTL allele of the WKY rat was different from that of the MNS rat; differing QTL alleles could have distinctive BP effects. Another possibility is that the WKY and MNS rats could have the same QTL allele in the D2N35/Nep region but that the larger substitution in the congenic S.MNS-Adh/D2Mit5 strain might contain other genes modifying its effect. The construction of S.MNS-Adh/D2Mit5 rats with a larger region of chromosome 2 was done because the putative QTL-containing region was poorly defined (larger) in the linkage analysis of the F₂ S×MNS population.² Also, there was evidence that a second QTL around At1b might exist,^{2 11 22} although At1b itself as the QTL was not supported by DNA sequence determinations of coding regions.¹¹ On the basis of previous data, one would expect the longer segment in S.MNS-Adh/D2Mit5 to lower BP more than the shorter segment of S.WKY-D2N35/Nep. The opposite was the case. Thus, the difference in BP between the congenic strains is unexplained, although each strain obviously had a major effect on BP.

The region between D2N35 and Nep on chromosome 2 harbors several genes that could be construed as candidates for the BP QTL. These include Camk, Nakα1, Gca, and Nep (Figure⇑). Because the substituted region is large (41 cM), speculation as to the identity of the actual QTL is premature.

Selected Abbreviations and Acronyms