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Wild rats (Rattus norvegicus) for mapping of disease-resistant genes
Experimental Animal Science
Wild Rats (Rattus norvegicus) for mapping of disease-resistant genes J. VAN DEN BRANDT, P. KovAcs, and I. KLOTING Department of Laboratory Animal Science at the Institute of Pathophysiology, University of Greifswald, Karlsburg, Germany
Summary Wild rat representing a disease-resistant phenotype and genotype, was used in a crossing study with spontaneously hypertensive rat (SHR) to search for quantitative trait loci (QTL) affecting blood pressure. Therefore, one male wild rat was crossed with SHR females and FI hybrids were transferred in a pathogen free environment by wet-hysterectomy and backcrossed onto hypertensive SHR rats resulting in first backcross hybrids (BC1). Considering that the F1 hybrids are not uniform, as are the cross hybrids of inbred rat strains, we selected 72 BC1 progeny of one F1 female, which were characterised for systolic blood pressure, measured by tail cuff method and were genetically analysed using 200 microsatellites covering the whole genome. We found suggestive linkage of blood pressure to region on chromosome 2 flanked by D2Mit8 and Fgg loci (lod score 2.3). In addition, possible interaction between genes on chromosomes 7 and 3, X and 3, 14 and 3, 13 and 11 was described, indicating that blood pressure development in the SHR might be the result of interacting genes.
Key words: SHR, QTL, hypertension, gene interaction, co-segregation analysis Inbreeding for many generations under more and more optimised environmental conditions has favoured the survival of disease alleles in inbred rat strains mostly without striking phenotypic consequences. Such "silent" disease genes may falsify results of cosegregation analysis searching for quantitative trait loci (QTLs) of complex characters as well as diseases, such as hypertension or diabetes mellitus. Wild rats, which should represent a disease-resistant phenotype and genotype may be an alternative for such cosegregation analysis. This idea prompted us to use normotensive wild rats and genetically hypertensive SHR/Mol rats to study QTLs of blood pressure and blood pressurerelated traits. One male wild rat was terminatively crossed with SHR females and the J. Exp. Anita. Sci. 2000; 41:57-60 Urban & Fischer Verlag hnp://www'urbanfischer'de/j°umals/jeansc 0939-8600/00/41/01-02-057 $12.00/0
58
J. VANDENBRANDT,P. KOVACSand I. KLOT~NG
(Wild x SHR) F1 hybrids were transferred in a pathogen free environment by wet-hysterectomy. F1 male and female rats were backcrossed onto hypertensive SHR rats resulting in first backcross hybrids (BC1). Considering that (Wild x SHR)F1 hybrids are not as uniform as cross hybrids of inbred rat strains attributed to the heterozygosity of wild rats which amounts to about 30% (KLOTIN6 et al. 1997), we selected 72 BC1 offspring of one F1 female for first phenotypic and genetic analysis. These BC1 hybrids were phenotypically characterised for systolic blood pressure between 12 and 14 weeks of age measured in conscious restrained animals on three separate time points between 9.00 and 11.00 a.m. by the tail-cuff method (Kent Scientific Corporation, Kent, England) as described recently in detail (KLOT[YGet al. 1995) and were used for genome-wide scan analysing 200 microsatellite markers on 20 autosomes and X chromosome covering more than 90% of the rat genome. The phenotypic characterisation of blood pressure in the parental rats (SHR: 196 _+ 10 mmHg; Wild rats 124 + 10 mmHg), F1 (125 + 10 mmHg) and BC1 hybrids (140 + 11 mmHg) clearly indicated dominance of low blood pressure of wild rats. Despite striking difference in blood pressure between wild and SHR rats (> 7SD), the genome-wide scan revealed no significant LOD score (>3.3) for blood pressure on any chromosome of these cross hybrids. As shown in Figure 1, there was only one suggestive linkage (LOD score
Chromosome 2 2.5Systolic
blood
I
,
2.0-
==
1.5-
o
o i/3 "1o
_o
05] !
1.0-
0.0
37.5 I
472 I'
22.4 I
4.3 2 B i
I
16 I
z8
32 t
8
[
?=
Fig. 1. Rat chromosome 2 linkage map and systolic blood pressure QTL localisation for male
((Wild x SHR)F1 x SHR) C1 hybrids.
J. VANDEN BRANDT,P. KovAcs and I. KLOTING
59
Table 1. Interacting genes of blood pressure in ((Wild x SHR)F1 x SHR) BC1 hybrids. Data given as mean _+SD. * SS-homozygous for alleles of the SHR rats. # SW-heterozygous for alleles of SHR and wild rats Loci
Lid (Chr. 7)
D3Mgh7 (Chr. 3)
SS* SS
SS SW#
DXWox28 (Chr. X)
D3Mgh7(Chr.3)
SS SS
SS SW
Ren (Chr. 13)
D10Mgh13 (Chr. 11)
SS SS
SS SW
Gck (Chr. 14)
D3Mghi6 (Chr. 3)
SS SW
SW SW
Blood pressure (mmHg)
p-value
150 +- 8 (13) 135 + 10 (16)
<.0001
145 +_ 10 (23) 133 + 9 (17)
<.0001
145 + 8 (13) 134 + 8 (18)
<.0001
146 + 8 (18) 133-+ 7 (15)
<.0001
> 1.9) found for blood pressure in males on chromosome 2 (LOD score 2.3 between D2Mit8 and Fgg), confirming earlier findings of others (CLARK et al. 1996). This negative balance may be explained by the dominant action of low blood pressure alleles of wild rats. Assuming that at least 2 alleles of the SHR rat are essential for hypertension, the dominance of low blood pressure of wild rats in one of the 2 essential hypertensive SHR alleles which will depress the hypertensive action of the 2nd SHR allele. Therefore, we searched for gene interaction to detect those alleles depressing and increasing blood pressure, respectively. As demonstrated in Table 1, homozygosity at loci D3Mgh7 on chromosome 3 and at Lid on chromosome 7 as well as at DXWox28 on chromosome X indicated significantly elevated blood pressure in comparison to those which were homozygous at Lid as well as DXWox28 but heterozygous at D3Mgh7 (p < .0001). In addition, significantly increased blood pressure was observed in homozygotes at loci Ren on chromosome 13 and DlOMghl3 located not only on chromosome 10 but on chromosome 11 in comparison with those which were homozygous at Ren and heterozygous at DlOMgh13 (145 _+8 vs. 134 + 8, p < .0001). In contrast to those gene interactions indicating that heterozygosity at one of two interacting blood pressure loci significantly decreased blood pressure, there was also an other relationships. Homozygosity at one locus, Gck on chromosome 14 and heterozygosity at the second locus D3Mgh16 on chromosome 3 showed significantly increased blood pressure, whereas, heterozygosity at both loci, Gck and D3Mghl6, significantly decreased the blood pressure (146 _+ 8 vs. 133 + 7, p < .0001). This finding indicates, that there are most probably genes in SHR rats which can also decrease blood pressure as previously described for loci on chromosomes 4 and 13 (ST. LEZIN et a1.1996, KovAcs et al. 1997), and that there are also genes in wild rats which contribute to increased blood pressure. These findings may be interpreted in
60
J. VANDEN BRANDT,e. KOVACSand I. KLOTING
the sense that normotension is the balance of interacting genes increasing and decreasing blood pressure which results in normotension and that a disturbance of this balance caused by mutations in one of the interacting genes can lead to hypertension and hypotension, respectively.
References CLARK,J.S., B. JEFFS,A.O. DAVIDSON,W.K. LEE, N.H. ANDERSON,M.T. BIHOREAU,M.J. BROSNAN, A.M. DEVLIN,A.W. KELMAN,K. LINDPAINTNER,and A.F. DOMINICZAK.1996. Quantitative trait loci in genetically hypertensive rats. Possible sex specificity. Hypertension 28: 898-906. KLOTING, I., M. STIELOW,and L. VOGT. 1995. Development of new animal models in diabetes research: spontaneously hypertensive-diabetic rats. Diabetes Res. 29, 1995, 127-138 KLOTINO, I., B. VOIGT, and P. KovKcs. 1997. Comparison of genetic variability at microsatellite loci in wild rats and inbred rat strains (Rattus norvegicus). Mamm. Genome 8:589-591. KOVACS, P., B. VOIGT, and I. KLOTING. 1997. Alleles of the spontaneously hypertensive rat decrease blood pressure at loci on chromosomes 4 and 13. Biochem. Biophys. Res. Commun. 238: 586-589. ST. LEZlN, E.M., M. PRAV~NE¢, A.L. WON6, W. LUI, N. WANG, S. Lu, H.J. JACOB, R.J. ROMAN, D.E. STEC, J.M. WANG, I.A. REID, and T.W. KURTZ. 1996. Effects of renin gene transfer on blood pressure and renin gene expression in a congenic strain of Dahl salt-resistant rats. J. Clin. Invest. 97: 522-527.
Corresponding author: J. VAN DEN BRANDT, Department of Laboratory Animal Science at the Institute of Pathophysiology, University of Greifswaid, D-17495 Karlsburg, Germany Tel.: +49 3834 8619152; Fax: +49 3834 8619111; e-mail: brandtj @mail.uni-greifswald.de
Wild Rats (Rattus norvegicus) for mapping of disease-resistant genes J. VAN DEN BRANDT, P. KovAcs, and I. KLOTING Department of Laboratory Animal Science at the Institute of Pathophysiology, University of Greifswald, Karlsburg, Germany
Summary Wild rat representing a disease-resistant phenotype and genotype, was used in a crossing study with spontaneously hypertensive rat (SHR) to search for quantitative trait loci (QTL) affecting blood pressure. Therefore, one male wild rat was crossed with SHR females and FI hybrids were transferred in a pathogen free environment by wet-hysterectomy and backcrossed onto hypertensive SHR rats resulting in first backcross hybrids (BC1). Considering that the F1 hybrids are not uniform, as are the cross hybrids of inbred rat strains, we selected 72 BC1 progeny of one F1 female, which were characterised for systolic blood pressure, measured by tail cuff method and were genetically analysed using 200 microsatellites covering the whole genome. We found suggestive linkage of blood pressure to region on chromosome 2 flanked by D2Mit8 and Fgg loci (lod score 2.3). In addition, possible interaction between genes on chromosomes 7 and 3, X and 3, 14 and 3, 13 and 11 was described, indicating that blood pressure development in the SHR might be the result of interacting genes.
Key words: SHR, QTL, hypertension, gene interaction, co-segregation analysis Inbreeding for many generations under more and more optimised environmental conditions has favoured the survival of disease alleles in inbred rat strains mostly without striking phenotypic consequences. Such "silent" disease genes may falsify results of cosegregation analysis searching for quantitative trait loci (QTLs) of complex characters as well as diseases, such as hypertension or diabetes mellitus. Wild rats, which should represent a disease-resistant phenotype and genotype may be an alternative for such cosegregation analysis. This idea prompted us to use normotensive wild rats and genetically hypertensive SHR/Mol rats to study QTLs of blood pressure and blood pressurerelated traits. One male wild rat was terminatively crossed with SHR females and the J. Exp. Anita. Sci. 2000; 41:57-60 Urban & Fischer Verlag hnp://www'urbanfischer'de/j°umals/jeansc 0939-8600/00/41/01-02-057 $12.00/0
58
J. VANDENBRANDT,P. KOVACSand I. KLOT~NG
(Wild x SHR) F1 hybrids were transferred in a pathogen free environment by wet-hysterectomy. F1 male and female rats were backcrossed onto hypertensive SHR rats resulting in first backcross hybrids (BC1). Considering that (Wild x SHR)F1 hybrids are not as uniform as cross hybrids of inbred rat strains attributed to the heterozygosity of wild rats which amounts to about 30% (KLOTIN6 et al. 1997), we selected 72 BC1 offspring of one F1 female for first phenotypic and genetic analysis. These BC1 hybrids were phenotypically characterised for systolic blood pressure between 12 and 14 weeks of age measured in conscious restrained animals on three separate time points between 9.00 and 11.00 a.m. by the tail-cuff method (Kent Scientific Corporation, Kent, England) as described recently in detail (KLOT[YGet al. 1995) and were used for genome-wide scan analysing 200 microsatellite markers on 20 autosomes and X chromosome covering more than 90% of the rat genome. The phenotypic characterisation of blood pressure in the parental rats (SHR: 196 _+ 10 mmHg; Wild rats 124 + 10 mmHg), F1 (125 + 10 mmHg) and BC1 hybrids (140 + 11 mmHg) clearly indicated dominance of low blood pressure of wild rats. Despite striking difference in blood pressure between wild and SHR rats (> 7SD), the genome-wide scan revealed no significant LOD score (>3.3) for blood pressure on any chromosome of these cross hybrids. As shown in Figure 1, there was only one suggestive linkage (LOD score
Chromosome 2 2.5Systolic
blood
I
,
2.0-
==
1.5-
o
o i/3 "1o
_o
05] !
1.0-
0.0
37.5 I
472 I'
22.4 I
4.3 2 B i
I
16 I
z8
32 t
8
[
?=
Fig. 1. Rat chromosome 2 linkage map and systolic blood pressure QTL localisation for male
((Wild x SHR)F1 x SHR) C1 hybrids.
J. VANDEN BRANDT,P. KovAcs and I. KLOTING
59
Table 1. Interacting genes of blood pressure in ((Wild x SHR)F1 x SHR) BC1 hybrids. Data given as mean _+SD. * SS-homozygous for alleles of the SHR rats. # SW-heterozygous for alleles of SHR and wild rats Loci
Lid (Chr. 7)
D3Mgh7 (Chr. 3)
SS* SS
SS SW#
DXWox28 (Chr. X)
D3Mgh7(Chr.3)
SS SS
SS SW
Ren (Chr. 13)
D10Mgh13 (Chr. 11)
SS SS
SS SW
Gck (Chr. 14)
D3Mghi6 (Chr. 3)
SS SW
SW SW
Blood pressure (mmHg)
p-value
150 +- 8 (13) 135 + 10 (16)
<.0001
145 +_ 10 (23) 133 + 9 (17)
<.0001
145 + 8 (13) 134 + 8 (18)
<.0001
146 + 8 (18) 133-+ 7 (15)
<.0001
> 1.9) found for blood pressure in males on chromosome 2 (LOD score 2.3 between D2Mit8 and Fgg), confirming earlier findings of others (CLARK et al. 1996). This negative balance may be explained by the dominant action of low blood pressure alleles of wild rats. Assuming that at least 2 alleles of the SHR rat are essential for hypertension, the dominance of low blood pressure of wild rats in one of the 2 essential hypertensive SHR alleles which will depress the hypertensive action of the 2nd SHR allele. Therefore, we searched for gene interaction to detect those alleles depressing and increasing blood pressure, respectively. As demonstrated in Table 1, homozygosity at loci D3Mgh7 on chromosome 3 and at Lid on chromosome 7 as well as at DXWox28 on chromosome X indicated significantly elevated blood pressure in comparison to those which were homozygous at Lid as well as DXWox28 but heterozygous at D3Mgh7 (p < .0001). In addition, significantly increased blood pressure was observed in homozygotes at loci Ren on chromosome 13 and DlOMghl3 located not only on chromosome 10 but on chromosome 11 in comparison with those which were homozygous at Ren and heterozygous at DlOMgh13 (145 _+8 vs. 134 + 8, p < .0001). In contrast to those gene interactions indicating that heterozygosity at one of two interacting blood pressure loci significantly decreased blood pressure, there was also an other relationships. Homozygosity at one locus, Gck on chromosome 14 and heterozygosity at the second locus D3Mgh16 on chromosome 3 showed significantly increased blood pressure, whereas, heterozygosity at both loci, Gck and D3Mghl6, significantly decreased the blood pressure (146 _+ 8 vs. 133 + 7, p < .0001). This finding indicates, that there are most probably genes in SHR rats which can also decrease blood pressure as previously described for loci on chromosomes 4 and 13 (ST. LEZIN et a1.1996, KovAcs et al. 1997), and that there are also genes in wild rats which contribute to increased blood pressure. These findings may be interpreted in
60
J. VANDEN BRANDT,e. KOVACSand I. KLOTING
the sense that normotension is the balance of interacting genes increasing and decreasing blood pressure which results in normotension and that a disturbance of this balance caused by mutations in one of the interacting genes can lead to hypertension and hypotension, respectively.
References CLARK,J.S., B. JEFFS,A.O. DAVIDSON,W.K. LEE, N.H. ANDERSON,M.T. BIHOREAU,M.J. BROSNAN, A.M. DEVLIN,A.W. KELMAN,K. LINDPAINTNER,and A.F. DOMINICZAK.1996. Quantitative trait loci in genetically hypertensive rats. Possible sex specificity. Hypertension 28: 898-906. KLOTING, I., M. STIELOW,and L. VOGT. 1995. Development of new animal models in diabetes research: spontaneously hypertensive-diabetic rats. Diabetes Res. 29, 1995, 127-138 KLOTINO, I., B. VOIGT, and P. KovKcs. 1997. Comparison of genetic variability at microsatellite loci in wild rats and inbred rat strains (Rattus norvegicus). Mamm. Genome 8:589-591. KOVACS, P., B. VOIGT, and I. KLOTING. 1997. Alleles of the spontaneously hypertensive rat decrease blood pressure at loci on chromosomes 4 and 13. Biochem. Biophys. Res. Commun. 238: 586-589. ST. LEZlN, E.M., M. PRAV~NE¢, A.L. WON6, W. LUI, N. WANG, S. Lu, H.J. JACOB, R.J. ROMAN, D.E. STEC, J.M. WANG, I.A. REID, and T.W. KURTZ. 1996. Effects of renin gene transfer on blood pressure and renin gene expression in a congenic strain of Dahl salt-resistant rats. J. Clin. Invest. 97: 522-527.
Corresponding author: J. VAN DEN BRANDT, Department of Laboratory Animal Science at the Institute of Pathophysiology, University of Greifswaid, D-17495 Karlsburg, Germany Tel.: +49 3834 8619152; Fax: +49 3834 8619111; e-mail: brandtj @mail.uni-greifswald.de