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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.
253, 204 –208 (1998)
RC989776
Genetic Analysis of Dystrophic Cardiac Calcification in DBA/2 Mice F. A. R. van den Broek,*,† R. Bakker,* M. den Bieman,† A. X. M. Fielmich-Bouwman,† A. G. Lemmens,† H. A. van Lith,† I. Nissen,‡ J. M. Ritskes-Hoitinga,‡ G. van Tintelen,* and L. F. M. van Zutphen† *Small Animal Facility, Agricultural University Wageningen, P.O. Box 8129, 6700 EV Wageningen, The Netherlands; †Department of Laboratory Animal Science, Faculty of Veterinary Medicine, Utrecht University, P.O. Box 80.166, 3508 TD Utrecht, The Netherlands; and ‡Biomedical Laboratory, Odense University, Winsloewparken 21, DK-5000 Odense C, Denmark
Received October 30, 1998
A DBA/2 3 D2B6F1 backcross was produced in order to study the genetic background of pathological soft tissue calcification in the mouse. Calcification was assessed in the myocardium, kidney and tongue. Significant co-segregation was found with the genotype of microsatellite markers on the proximal end of Chromosome 7. This region contains a candidate gene, Hrc, coding for the histidine-rich calcium binding protein in the sarcoplasmatic reticulum. The results support the hypothesis that the gene previously reported to be responsible for DCC (dystrophic cardiac calcification) in C3H mice (1) causes generalized soft tissue calcification in DBA/2 mice. © 1998 Academic Press
Eaton et al. (2) described dystrophic cardiac calcification (DCC) in several strains of laboratory mice. Depending on the strain, the myocardium or the epicardium was affected. QTL analysis of both an F2intercross and a set of recombinant inbred strains revealed that in the C3H strain a gene on Chromosome 7, tentatively named Dyscalc, is responsible for myocardial calcification that is observed in this strain (1). In the same study, suggestive linkage of calcification with microsatellite markers on Chromosomes 4, 8 and 12 was also observed. In DBA/2 mice, cardiac calcification is not limited to the myocardium, but also involves the epicardium. Besides the heart, the kidney and the tongue are frequently calcified (3) and, to a lesser extent, many other organs. In the present study we tested whether or not the generalized soft tissue calcification in DBA/2 mice and the myocardial calcification in C3H mice have the same genetic background. In addition, the plasma parathyroid hormone (PTH) level was mea0006-291X/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.
sured in order to study the role of this hormone in the calcification process. MATERIALS AND METHODS Animals, housing, and diet. Male DBA/2Ola and female D2B6F1 (DBA/2Ola 3 C57BL/6Ola) mice, 15 of each, were purchased from Harlan Netherlands B.V. (Horst, The Netherlands) when aged five weeks. At the age of nine weeks they were mated on a one male-one female basis; the male mice were removed after 5-6 weeks. Out of these 15 combinations fourteen nests were born. This DDB backcross offspring (n5112) was weaned at the age of three weeks. The backcross offspring was housed after weaning in groups of three of the same sex in Macrolon Type II cages. After breeding the male and female parents were housed individually, again in Macrolon Type II cages. All mice had free access to drinking water and food pellets. For the parents and the backcross animals until the age of four weeks this was a commercial diet (RMH-B, Hope Farms B.V., Woerden, The Netherlands). Room temperature and humidity were controlled (2022°C and 40-60%, respectively); the artificial daylight period lasted from 6.00 until 18.00h. At the age of four weeks, the backcross mice were changed to a high-phosphorus semipurified diet (0.8% w/w), that has previously been shown to enhance soft tissue calcification in DBA/2 mice (4). The mice remained on this diet until dissection, three weeks later. Blood sampling and tissue collection. The mice were killed by exsanguination via orbital puncture, while under ether anaesthesia. For DNA analysis the liver was excised and cut in halves. Each half was put into a separate vial and deep-frozen in liquid nitrogen. Blood plasma was used for the determination of parathyroid hormone (PTH), calcium, magnesium and phosphorus concentrations. The heart was excised for histological examination. The kidney and tongue were scored either calcification negative (0) or positive (1) macroscopically. A kidney was excised and deep-frozen until chemical analysis. Chemical analysis. The deep-frozen kidneys were dried (106°C, 16h) and ashed (500°C, 17h). The ash was dissolved in 6M HCl and calcium and magnesium were measured by atomic absorption spectrophotometry on a Varian-AA275 (Varian, Springville, Australia). Phosphorus was measured using a commercial, Phos-Ultimate 7 kit on a Cobas-BIO automatic analyser (Roche Diagnostic Systems, Hoffmann-La Roche, Basel, Switzerland). Minerals in plasma were
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Vol. 253, No. 2, 1998
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS TABLE 1
Criteria Used to Assess the Degree of Calcification in Heart Score
Microscopic necropsy findings
0
No calcium deposits One or few small deposits per slide Many small or a few large foci per slide Slide scattered with foci or confluencing foci affecting a large part of the slide
Note. Heart scores are averaged for 9 –10 slides.
analysed without sample pretreatment. Kidney mineral concentrations are expressed as mmol mineral/g dry weight. PTH was measured with a commercially available rat PTH IRMA kit (Rat-PTH, Nichols Institute, Nijmegen, The Netherlands), that was previously validated for mice (5). A Packard Multi Prias gamma counter (Packard, Downers Grove, Illinois) was used to assess radioactivity. Histological analysis. After storage in 4% buffered formalin, the heart samples were cut transversely into 4mm slides. Slides were picked at even 70mm intervals and HE stained. Thus per animal 9 –16 slides of the heart were collected. Scores were given to each slide, separately for the myocardium and the epicardium, according to the criteria listed in Table 1; the multiple scores for each animal were averaged to obtain a representative DCC severity score. Ten slides were examined, unless these were all negative, in which case all slides were examined. In three cases only nine slides were available. Genetic typing by PCR. DNA was isolated from the liver samples using a standard isolation method (6). DNA was resuspended in a 10mM Tris, 0.2mM EDTA, pH 8.0 solution. The DNA concentrations were determined by measuring the A260. Markers, polymorphic between the DBA/2 and C57BL/6 strains, were selected after consultation of the MIT mouse genome database through the internet (http//www.genome.wi.mit.edu). Primer pairs were purchased from Research Genetics (Huntsville, AL), and PCRs were performed according to the manufacturer’s protocol. All animals were genotyped for the following 21 markers: D12Mit38, D4Mit142, D4Mit338, D8Mit124, D8Mit140, D8Mit113, D8Mit271, D7Mit178, D7Mit57, D7Mit117, D7Mit308, D7Mit270, D7Mit229, D7Mit230, D7Mit82, D7Mit297, D7Mit147, D7Mit31, D7Mit281, D7Mit330 and D7Mit333. PCR products were visualized by ethidium bromide staining after agarose gel electrophoresis (3% Pronarose MS-8, Hispanagar).
Subsequently the Student’s t-test was used to evaluate the differences in the means of these two transformed traits between homozygotes and heterozygotes at each of the Chromosome 4, 8 and 12 marker loci. Suggestive and significant linkage were assumed when p#0.0034 and p#0.0001, respectively (9). QTLs affecting the traits under study were mapped relatively to the Chromosome 7 markers with the use of the MapQTL version 3.0 programme (10). For normally distributed traits the interval mapping module was used (11), whereas for non-normally distributed traits the non-parametric mapping module was used (12). Results were expressed as LOD-scores for normally distributed traits and Kruskal-Wallis test statistics for non-normally distributed traits. The LOD-score threshold for significant linkage was preset at 3.3 and for suggestive linkage at 1.9 (9). For the non-parametric approach p#0.05 and p#0.005 were considered to indicate suggestive and significant linkage, respectively (10).
RESULTS Calcifications Histological examination revealed that in 61 out of 112 backcross animals myocardial calcification was present (35 females and 26 males). Macroscopical screening of the kidneys revealed calcification in 58 animals (33 females and 25 males), whereas in 46 animals (24 females and 22 males) tongue calcifications were found. In total 41 females (73 per cent) and 35 males (63 per cent) had calcific lesions in one or more of the investigated organs. The chemically determined kidney calcium content corresponded well with the macroscopically observed kidney calcification. Females with positive macroscopy scores had an average calcium content of 11066689 mmol/g dry weight, compared to 250 6 260 mmol/g dry weight for their macroscopy-negative counterparts (means6SD, p,0.001 in the two-tailed unpaired Student’s t-test on transformed data). Also, macroscopically positive male mice had significantly higher (p,0.01) in the two-tailed unpaired Student’s t-test on transformed data) kidney calcium contents than nega-
Map construction and QTL analysis. Inspection of the segregation ratios for the individual markers on Chromosome 7, by means of the x2 goodness-of-fit test, revealed that none of the markers had a significant segregation distortion. The genetic map distance for the Chromosome 7 markers was computed with the programme JoinMap, version 2.0 (7,8). The recombination frequencies were converted to map distances in cM with the Kosambi function. The map thus obtained is given in Table 2. Co-segregation of the individual Chromosome 4, 8 and 12 markers to dichotomous traits was assessed by applying the x2-test of independence with the Yates’ improved approximation of the x2distribution, using a 2 3 2 contingency table. Statistical significance of differences of ordinal traits between homozygote and heterozygote marker loci at Chromosome 4, 8 and 12 was evaluated by the MannWhitney U-test. Before statistical analysis the kidney calcium content and plasma PTH concentration were transformed to a normal distribution, using the following transformation: y510Log(x1c), where y is the transformed trait value, x is the original trait value and c is a constant. 205
TABLE 2
Marker Location Name
cM
D7Mit178 D7Mit57 D7Mit117 D7Mit308 D7Mit270 D7Mit229 D7Mit230 D7Mit82 D7Mit297 D7Mit147 D7Mit31 D7Mit281 D7Mit330 D7Mit333
0.0 9.4 15.1 16.0 24.4 26.2 27.1 29.8 32.5 39.2 45.0 60.9 65.6 83.8
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tive males (929 6 716 and 45 6137 mmol/g dry weight, respectively; means 6 SD). Both in males and females, the plasma PTH correlated significantly with the kidney calcium content (R50.68 and R50.70, respectively) and to a lesser extent also with the myocardial score (R50.57 and R50.31 respectively), both according to the Spearman rank correlation test (p,0.01). Genetic Analysis Polymorphism was established for all fourteen Chromosome 7 microsatellites in the backcross and parental animals. Figures 1 and 2 represent the Kruskal-Wallis test statistics and LOD-scores, respectively, for Chromosome 7. The findings for male and female mice are presented separately. In female mice highly significant Kruskal-Wallis test statistics were observed in the 18-39cM region of Chromosome 7 regarding the morphological calcification scores in kidney, tongue and myocardium (Fig. 1a). In males the calcification of the myocardium and the tongue resulted in high Kruskal-Wallis test statistics, with the highest scores in the region between 10 and 30 cM (Fig. 1b). For the macroscopically established kidney calcification in males, the Kruskal-Wallis test statistics revealed less conclusive results, but statistical significance was revealed in the region between 10 and 40 cM. For female mice, LOD-scores obtained for plasma PTH and kidney calcium content, after a logarithmic transformation, were statistically significant (Fig. 2a). More than 45 per cent of the biological variation of kidney calcium content in female mice could be attributed to gene-effects of a QTL in this region. In males suggestive LOD-scores for plasma PTH were observed (Fig. 2b). For the kidney calcium content two LOD-score peaks reached the significant linkage threshold. About 25 per cent of the biological variation of the kidney calcium content could be attributed to the gene-effects of a QTL in this region. Neither epicardial calcification nor the plasma parameters magnesium, phosphorus and calcium showed significant association with Chromosome 7 markers in either sex. Also, no suggestive linkage was observed for any of the traits under study with markers on markers on Chromosomes 4, 8 and 12. DISCUSSION QTL analysis of an F2 population and, derived from the DCC susceptible C3H inbred strain, has indicated that a gene on the proximal end of Chromosome 7, tentatively indicated as Dyscalc, might be responsible for the myocardial calcification in C3H mice (1). Recently, evidence was presented that in STS mice the
FIG. 1. Kruskal–Wallis test statistics for the morphological scores regarding heart, kidney, and tongue calcification on chromosome 7, in female (a) and male (b) DDB-backcross mice on chromosome 7. Heavy graph lines represent tongue calcification, thin graph lines heart calcification, and intermediate graph lines kidney calcification. The horizontal continuous and dotted lines depict significant and suggestive linkage, respectively. The most likely position of the Hrc gene is indicated by an arrow head. Dots on the X-axis represent the positions of microsatellite markers (see Table 2).
same gene might be responsible for generalized soft tissue calcification (13). In the present DDB backcross, calcification of heart, tongue and kidney were also found to be significantly associated with markers on the proximal region of Chromosome 7 (Figs. 1 and 2). These results seem to support the hypothesis that calcification of the myocardium, tongue and kidney have a common genetic basis. The LOD-scores and Kruskal-Wallis test statistics for heart, tongue and kidney calcification in the female mice (Fig. 1a and 2a) are in line with the LOD-scores observed for myocardial calcification in female C3H mice (1). The current findings in male mice, however, (Fig. 1b and 2b) suggest that more than one QTL is responsible for the observed variation in soft tissue calcification. Earlier reports suggested that Chromosome 7 contains a major gene involved in DCC (1,14), and that regions on Chromosomes 4 (1,14), 8 and 12 (1) might contain loci that play a minor role in the calcification process. However, besides a major gene on Chromo-
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FIG. 2. LOD scores for chemically analysed kidney calcium content and plasma PTH concentration in female (a) and male (b) DDB-backcross mice on chromosome 7. Heavy graph lines represent plasma PTH and thin graph lines kidney calcium content. Peaks with significant linkage are cut off at 61 LOD-score. The horizontal continuous and dotted lines depict significant and suggestive linkage, respectively. The most likely position of the Hrc gene is indicated by an arrow. Dots on the X-axis represent position of microsatellite markers (see Table 2).
some 7, we did not find evidence for the involvement of genes located in the previously suggested regions in the present study. The high percentage of biological variation that could be attributed to gene-effects of a QTL on the proximal Chromosome 7 suggests that this region contains the major gene for soft tissue calcification, except for in the epicardium. To exaggerate the gene effect Ivandic et al. (1) have used a high-fat, high-cholesterol diet. Van den Broek and Beynen (15) have shown that a high-fat diet did not enhance DCC in DBA/2 mice. Recently it has also been shown that in DBA/2 mice cholesterol absorption from high-cholesterol diets is less efficient than in C3H mice (16). Van den Broek and Beynen (4) have shown that a high-phosphorus diet leads to progressed DCC in DBA/2 mice. Therefore, this diet was used in the present study to enhance soft tissue calcification. A high phosphorus intake may influence plasma PTH. In the present study plasma PTH concentrations were high in association with heart- and kidney calci-
fication, but the co-segregation with microsatellite markers on Chromosome 7 was less evident. This suggests that a high PTH-response to dietary phosphorus is probably not the cause of calcification. Instead, PTH may have risen as a result of calcification-induced kidney failure (secondary hyperparathyreoidy). Furthermore, for the plasma minerals no relation with calcification could be observed. Thus, systemically originated metastatic calcification is probably not the cause of DCC. Yet, DCC seems to be associated with an abnormal calcium metabolism (3). Our findings of generalized soft tissue calcification suggest that the effect of the putative gene Dyscalc is not limited to the heart. Hrc, a gene coding for the histidine-rich calcium binding protein (HRC) in the sarcoplasmatic reticulum (SR), is positioned in the middle of the region that correlates with myocardial calcification in C3H (1). Preliminary results suggest a similar correlation in DBA/2 mice (14). Now it is shown that Hrc in DBA/2 mice is positioned in the middle of a region of Chromosome 7 that is strongly associated with generalized soft tissue calcification (Figures 1 and 2). Given its properties and location within the SR, an aberrant HRC production will affect cell calcium homeostasis. A resulting defect SR function could result in an increased fluctuation or average concentration of ionized calcium within the muscle cell, which in turn might deregulate the function of mitochondria. Indeed calcification in DCC starts in mitochondria (17) coinciding with abnormal ultrastructural features of the sarcoplasmatic reticulum (18). Thus it is tempting to speculate that the putative gene Dyscalc is identical to the candidate gene Hrc. In vitro studies on the calcium binding activity of HRC from mice differing in susceptibility towards soft tissue calcification might further clarify the role of this gene. REFERENCES
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1. Ivandic, B. T., Qiao, J. H., Machleder, D., Liao, F., Drake, T. A., and Lusis, A. J. (1996) Proc. Natl. Acad. Sci. USA 93, 5483–5488. 2. Eaton, G. J., Custer, R. P., Johnson, F. N., and Stabenow, K. T. (1978) Am. J. Pathol. 90, 173–186. 3. Van den Broek., F. A. R., Beems, R. B., Van Tintelen, G., Lemmens, A. G., Fielmich-Bouwman, A. X. M., and Beynen, A. C. (1997) Lab. Anim. 31, 74 – 80. 4. Van den Broek, F. A. R., and Beynen, A. C. (1998) Lab. Anim. 32, 483– 491. 5. Meyer, R. A., Morgan, P. L., and Meyer, M. H. (1994) Endocrine 2, 1127–1132. 6. Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K. (1987) Current Protocols in Molecular Biology, Green Publishing Assoc. Wiley–InterScience, New York. 7. Stam, P., (1993) Plant J. 3, 739 –744. 8. Stam, P., and Van Ooijen, J. W., (1995) Join Map Version 2.0. Software for the Calculation of Genetic Linkage Maps. CPRO– DLO, Wageningen.
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9. Lander, E. S., and Kruglyak, L. (1995) Nat. Genet. 11, 241– 247. 10. Van Ooijen, J. W., and Maliepaard, C. (1996) MapQTL version 3.0: Software for the Calculation of QTL Positions on Genetic Maps. CPRO–DLO, Wageningen. 11. Lander, E. S., and Botstein (1989) Genetics 121, 185–199. 12. Kruglyak, L., and Lander, E. S. (1995) Genetics 139, 1421– 1428. 13. Van den Broek, F. A. R. (1998) Dystrophic Cardiac Calcification in Laboratory Mice, thesis, Utrecht University, Utrecht.
14. Brunnert, S. R., Shi, S., and Chang, B. (1998) Contemp. Top. 37, 95. 15. Van den Broek, F. A. R., and Beynen, A. C. (1998) Lab. Anim. 32, 206 –213. 16. Carter, C. P., Howles, P. N., and Hui, D. Y. (1997) J. Nutr. 127, 1344 –1348. 17. Van Vleet, J. F., and Ferrans, V. J. (1987) Am. J. Vet. Res. 48, 255–261. 18. Itagaki, S., Maeda, N., Doi, K., and Mitsuoka, T. (1988) Vet. Pathol. 25, 393–395.
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RC989776
Genetic Analysis of Dystrophic Cardiac Calcification in DBA/2 Mice F. A. R. van den Broek,*,† R. Bakker,* M. den Bieman,† A. X. M. Fielmich-Bouwman,† A. G. Lemmens,† H. A. van Lith,† I. Nissen,‡ J. M. Ritskes-Hoitinga,‡ G. van Tintelen,* and L. F. M. van Zutphen† *Small Animal Facility, Agricultural University Wageningen, P.O. Box 8129, 6700 EV Wageningen, The Netherlands; †Department of Laboratory Animal Science, Faculty of Veterinary Medicine, Utrecht University, P.O. Box 80.166, 3508 TD Utrecht, The Netherlands; and ‡Biomedical Laboratory, Odense University, Winsloewparken 21, DK-5000 Odense C, Denmark
Received October 30, 1998
A DBA/2 3 D2B6F1 backcross was produced in order to study the genetic background of pathological soft tissue calcification in the mouse. Calcification was assessed in the myocardium, kidney and tongue. Significant co-segregation was found with the genotype of microsatellite markers on the proximal end of Chromosome 7. This region contains a candidate gene, Hrc, coding for the histidine-rich calcium binding protein in the sarcoplasmatic reticulum. The results support the hypothesis that the gene previously reported to be responsible for DCC (dystrophic cardiac calcification) in C3H mice (1) causes generalized soft tissue calcification in DBA/2 mice. © 1998 Academic Press
Eaton et al. (2) described dystrophic cardiac calcification (DCC) in several strains of laboratory mice. Depending on the strain, the myocardium or the epicardium was affected. QTL analysis of both an F2intercross and a set of recombinant inbred strains revealed that in the C3H strain a gene on Chromosome 7, tentatively named Dyscalc, is responsible for myocardial calcification that is observed in this strain (1). In the same study, suggestive linkage of calcification with microsatellite markers on Chromosomes 4, 8 and 12 was also observed. In DBA/2 mice, cardiac calcification is not limited to the myocardium, but also involves the epicardium. Besides the heart, the kidney and the tongue are frequently calcified (3) and, to a lesser extent, many other organs. In the present study we tested whether or not the generalized soft tissue calcification in DBA/2 mice and the myocardial calcification in C3H mice have the same genetic background. In addition, the plasma parathyroid hormone (PTH) level was mea0006-291X/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.
sured in order to study the role of this hormone in the calcification process. MATERIALS AND METHODS Animals, housing, and diet. Male DBA/2Ola and female D2B6F1 (DBA/2Ola 3 C57BL/6Ola) mice, 15 of each, were purchased from Harlan Netherlands B.V. (Horst, The Netherlands) when aged five weeks. At the age of nine weeks they were mated on a one male-one female basis; the male mice were removed after 5-6 weeks. Out of these 15 combinations fourteen nests were born. This DDB backcross offspring (n5112) was weaned at the age of three weeks. The backcross offspring was housed after weaning in groups of three of the same sex in Macrolon Type II cages. After breeding the male and female parents were housed individually, again in Macrolon Type II cages. All mice had free access to drinking water and food pellets. For the parents and the backcross animals until the age of four weeks this was a commercial diet (RMH-B, Hope Farms B.V., Woerden, The Netherlands). Room temperature and humidity were controlled (2022°C and 40-60%, respectively); the artificial daylight period lasted from 6.00 until 18.00h. At the age of four weeks, the backcross mice were changed to a high-phosphorus semipurified diet (0.8% w/w), that has previously been shown to enhance soft tissue calcification in DBA/2 mice (4). The mice remained on this diet until dissection, three weeks later. Blood sampling and tissue collection. The mice were killed by exsanguination via orbital puncture, while under ether anaesthesia. For DNA analysis the liver was excised and cut in halves. Each half was put into a separate vial and deep-frozen in liquid nitrogen. Blood plasma was used for the determination of parathyroid hormone (PTH), calcium, magnesium and phosphorus concentrations. The heart was excised for histological examination. The kidney and tongue were scored either calcification negative (0) or positive (1) macroscopically. A kidney was excised and deep-frozen until chemical analysis. Chemical analysis. The deep-frozen kidneys were dried (106°C, 16h) and ashed (500°C, 17h). The ash was dissolved in 6M HCl and calcium and magnesium were measured by atomic absorption spectrophotometry on a Varian-AA275 (Varian, Springville, Australia). Phosphorus was measured using a commercial, Phos-Ultimate 7 kit on a Cobas-BIO automatic analyser (Roche Diagnostic Systems, Hoffmann-La Roche, Basel, Switzerland). Minerals in plasma were
204
Vol. 253, No. 2, 1998
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS TABLE 1
Criteria Used to Assess the Degree of Calcification in Heart Score
Microscopic necropsy findings
0
No calcium deposits One or few small deposits per slide Many small or a few large foci per slide Slide scattered with foci or confluencing foci affecting a large part of the slide
Note. Heart scores are averaged for 9 –10 slides.
analysed without sample pretreatment. Kidney mineral concentrations are expressed as mmol mineral/g dry weight. PTH was measured with a commercially available rat PTH IRMA kit (Rat-PTH, Nichols Institute, Nijmegen, The Netherlands), that was previously validated for mice (5). A Packard Multi Prias gamma counter (Packard, Downers Grove, Illinois) was used to assess radioactivity. Histological analysis. After storage in 4% buffered formalin, the heart samples were cut transversely into 4mm slides. Slides were picked at even 70mm intervals and HE stained. Thus per animal 9 –16 slides of the heart were collected. Scores were given to each slide, separately for the myocardium and the epicardium, according to the criteria listed in Table 1; the multiple scores for each animal were averaged to obtain a representative DCC severity score. Ten slides were examined, unless these were all negative, in which case all slides were examined. In three cases only nine slides were available. Genetic typing by PCR. DNA was isolated from the liver samples using a standard isolation method (6). DNA was resuspended in a 10mM Tris, 0.2mM EDTA, pH 8.0 solution. The DNA concentrations were determined by measuring the A260. Markers, polymorphic between the DBA/2 and C57BL/6 strains, were selected after consultation of the MIT mouse genome database through the internet (http//www.genome.wi.mit.edu). Primer pairs were purchased from Research Genetics (Huntsville, AL), and PCRs were performed according to the manufacturer’s protocol. All animals were genotyped for the following 21 markers: D12Mit38, D4Mit142, D4Mit338, D8Mit124, D8Mit140, D8Mit113, D8Mit271, D7Mit178, D7Mit57, D7Mit117, D7Mit308, D7Mit270, D7Mit229, D7Mit230, D7Mit82, D7Mit297, D7Mit147, D7Mit31, D7Mit281, D7Mit330 and D7Mit333. PCR products were visualized by ethidium bromide staining after agarose gel electrophoresis (3% Pronarose MS-8, Hispanagar).
Subsequently the Student’s t-test was used to evaluate the differences in the means of these two transformed traits between homozygotes and heterozygotes at each of the Chromosome 4, 8 and 12 marker loci. Suggestive and significant linkage were assumed when p#0.0034 and p#0.0001, respectively (9). QTLs affecting the traits under study were mapped relatively to the Chromosome 7 markers with the use of the MapQTL version 3.0 programme (10). For normally distributed traits the interval mapping module was used (11), whereas for non-normally distributed traits the non-parametric mapping module was used (12). Results were expressed as LOD-scores for normally distributed traits and Kruskal-Wallis test statistics for non-normally distributed traits. The LOD-score threshold for significant linkage was preset at 3.3 and for suggestive linkage at 1.9 (9). For the non-parametric approach p#0.05 and p#0.005 were considered to indicate suggestive and significant linkage, respectively (10).
RESULTS Calcifications Histological examination revealed that in 61 out of 112 backcross animals myocardial calcification was present (35 females and 26 males). Macroscopical screening of the kidneys revealed calcification in 58 animals (33 females and 25 males), whereas in 46 animals (24 females and 22 males) tongue calcifications were found. In total 41 females (73 per cent) and 35 males (63 per cent) had calcific lesions in one or more of the investigated organs. The chemically determined kidney calcium content corresponded well with the macroscopically observed kidney calcification. Females with positive macroscopy scores had an average calcium content of 11066689 mmol/g dry weight, compared to 250 6 260 mmol/g dry weight for their macroscopy-negative counterparts (means6SD, p,0.001 in the two-tailed unpaired Student’s t-test on transformed data). Also, macroscopically positive male mice had significantly higher (p,0.01) in the two-tailed unpaired Student’s t-test on transformed data) kidney calcium contents than nega-
Map construction and QTL analysis. Inspection of the segregation ratios for the individual markers on Chromosome 7, by means of the x2 goodness-of-fit test, revealed that none of the markers had a significant segregation distortion. The genetic map distance for the Chromosome 7 markers was computed with the programme JoinMap, version 2.0 (7,8). The recombination frequencies were converted to map distances in cM with the Kosambi function. The map thus obtained is given in Table 2. Co-segregation of the individual Chromosome 4, 8 and 12 markers to dichotomous traits was assessed by applying the x2-test of independence with the Yates’ improved approximation of the x2distribution, using a 2 3 2 contingency table. Statistical significance of differences of ordinal traits between homozygote and heterozygote marker loci at Chromosome 4, 8 and 12 was evaluated by the MannWhitney U-test. Before statistical analysis the kidney calcium content and plasma PTH concentration were transformed to a normal distribution, using the following transformation: y510Log(x1c), where y is the transformed trait value, x is the original trait value and c is a constant. 205
TABLE 2
Marker Location Name
cM
D7Mit178 D7Mit57 D7Mit117 D7Mit308 D7Mit270 D7Mit229 D7Mit230 D7Mit82 D7Mit297 D7Mit147 D7Mit31 D7Mit281 D7Mit330 D7Mit333
0.0 9.4 15.1 16.0 24.4 26.2 27.1 29.8 32.5 39.2 45.0 60.9 65.6 83.8
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tive males (929 6 716 and 45 6137 mmol/g dry weight, respectively; means 6 SD). Both in males and females, the plasma PTH correlated significantly with the kidney calcium content (R50.68 and R50.70, respectively) and to a lesser extent also with the myocardial score (R50.57 and R50.31 respectively), both according to the Spearman rank correlation test (p,0.01). Genetic Analysis Polymorphism was established for all fourteen Chromosome 7 microsatellites in the backcross and parental animals. Figures 1 and 2 represent the Kruskal-Wallis test statistics and LOD-scores, respectively, for Chromosome 7. The findings for male and female mice are presented separately. In female mice highly significant Kruskal-Wallis test statistics were observed in the 18-39cM region of Chromosome 7 regarding the morphological calcification scores in kidney, tongue and myocardium (Fig. 1a). In males the calcification of the myocardium and the tongue resulted in high Kruskal-Wallis test statistics, with the highest scores in the region between 10 and 30 cM (Fig. 1b). For the macroscopically established kidney calcification in males, the Kruskal-Wallis test statistics revealed less conclusive results, but statistical significance was revealed in the region between 10 and 40 cM. For female mice, LOD-scores obtained for plasma PTH and kidney calcium content, after a logarithmic transformation, were statistically significant (Fig. 2a). More than 45 per cent of the biological variation of kidney calcium content in female mice could be attributed to gene-effects of a QTL in this region. In males suggestive LOD-scores for plasma PTH were observed (Fig. 2b). For the kidney calcium content two LOD-score peaks reached the significant linkage threshold. About 25 per cent of the biological variation of the kidney calcium content could be attributed to the gene-effects of a QTL in this region. Neither epicardial calcification nor the plasma parameters magnesium, phosphorus and calcium showed significant association with Chromosome 7 markers in either sex. Also, no suggestive linkage was observed for any of the traits under study with markers on markers on Chromosomes 4, 8 and 12. DISCUSSION QTL analysis of an F2 population and, derived from the DCC susceptible C3H inbred strain, has indicated that a gene on the proximal end of Chromosome 7, tentatively indicated as Dyscalc, might be responsible for the myocardial calcification in C3H mice (1). Recently, evidence was presented that in STS mice the
FIG. 1. Kruskal–Wallis test statistics for the morphological scores regarding heart, kidney, and tongue calcification on chromosome 7, in female (a) and male (b) DDB-backcross mice on chromosome 7. Heavy graph lines represent tongue calcification, thin graph lines heart calcification, and intermediate graph lines kidney calcification. The horizontal continuous and dotted lines depict significant and suggestive linkage, respectively. The most likely position of the Hrc gene is indicated by an arrow head. Dots on the X-axis represent the positions of microsatellite markers (see Table 2).
same gene might be responsible for generalized soft tissue calcification (13). In the present DDB backcross, calcification of heart, tongue and kidney were also found to be significantly associated with markers on the proximal region of Chromosome 7 (Figs. 1 and 2). These results seem to support the hypothesis that calcification of the myocardium, tongue and kidney have a common genetic basis. The LOD-scores and Kruskal-Wallis test statistics for heart, tongue and kidney calcification in the female mice (Fig. 1a and 2a) are in line with the LOD-scores observed for myocardial calcification in female C3H mice (1). The current findings in male mice, however, (Fig. 1b and 2b) suggest that more than one QTL is responsible for the observed variation in soft tissue calcification. Earlier reports suggested that Chromosome 7 contains a major gene involved in DCC (1,14), and that regions on Chromosomes 4 (1,14), 8 and 12 (1) might contain loci that play a minor role in the calcification process. However, besides a major gene on Chromo-
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FIG. 2. LOD scores for chemically analysed kidney calcium content and plasma PTH concentration in female (a) and male (b) DDB-backcross mice on chromosome 7. Heavy graph lines represent plasma PTH and thin graph lines kidney calcium content. Peaks with significant linkage are cut off at 61 LOD-score. The horizontal continuous and dotted lines depict significant and suggestive linkage, respectively. The most likely position of the Hrc gene is indicated by an arrow. Dots on the X-axis represent position of microsatellite markers (see Table 2).
some 7, we did not find evidence for the involvement of genes located in the previously suggested regions in the present study. The high percentage of biological variation that could be attributed to gene-effects of a QTL on the proximal Chromosome 7 suggests that this region contains the major gene for soft tissue calcification, except for in the epicardium. To exaggerate the gene effect Ivandic et al. (1) have used a high-fat, high-cholesterol diet. Van den Broek and Beynen (15) have shown that a high-fat diet did not enhance DCC in DBA/2 mice. Recently it has also been shown that in DBA/2 mice cholesterol absorption from high-cholesterol diets is less efficient than in C3H mice (16). Van den Broek and Beynen (4) have shown that a high-phosphorus diet leads to progressed DCC in DBA/2 mice. Therefore, this diet was used in the present study to enhance soft tissue calcification. A high phosphorus intake may influence plasma PTH. In the present study plasma PTH concentrations were high in association with heart- and kidney calci-
fication, but the co-segregation with microsatellite markers on Chromosome 7 was less evident. This suggests that a high PTH-response to dietary phosphorus is probably not the cause of calcification. Instead, PTH may have risen as a result of calcification-induced kidney failure (secondary hyperparathyreoidy). Furthermore, for the plasma minerals no relation with calcification could be observed. Thus, systemically originated metastatic calcification is probably not the cause of DCC. Yet, DCC seems to be associated with an abnormal calcium metabolism (3). Our findings of generalized soft tissue calcification suggest that the effect of the putative gene Dyscalc is not limited to the heart. Hrc, a gene coding for the histidine-rich calcium binding protein (HRC) in the sarcoplasmatic reticulum (SR), is positioned in the middle of the region that correlates with myocardial calcification in C3H (1). Preliminary results suggest a similar correlation in DBA/2 mice (14). Now it is shown that Hrc in DBA/2 mice is positioned in the middle of a region of Chromosome 7 that is strongly associated with generalized soft tissue calcification (Figures 1 and 2). Given its properties and location within the SR, an aberrant HRC production will affect cell calcium homeostasis. A resulting defect SR function could result in an increased fluctuation or average concentration of ionized calcium within the muscle cell, which in turn might deregulate the function of mitochondria. Indeed calcification in DCC starts in mitochondria (17) coinciding with abnormal ultrastructural features of the sarcoplasmatic reticulum (18). Thus it is tempting to speculate that the putative gene Dyscalc is identical to the candidate gene Hrc. In vitro studies on the calcium binding activity of HRC from mice differing in susceptibility towards soft tissue calcification might further clarify the role of this gene. REFERENCES
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