Mapping of microsatellite loci and association of aorta atherosclerosis with LG VI markers in the rabbit
R. KORSTANJE,
G. F. GILLISSEN,
L. P. KODDE,
M. DEN BIEMAN,
A. LANKHORST,
L. F. M. VAN ZUTPHEN and
H. A. VAN LITH
Department of Laboratory Animal Science, Faculty of Veterinary Medicine, Utrecht University, 3508 TD Utrecht, The Netherlands
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ABSTRACT
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Twenty-three rabbit microsatellites were extracted from the EMBL nucleotide database. Nine of these markers, together with nine earlier published microsatellite markers, were found to be polymorphic between the AX/JU and IIIVO/JU inbred strains. By using an F2 intercross we could integrate five markers into the rabbit linkage map. One anonymous microsatellite marker could be assigned to chromosome 1, and one microsatellite marker, located within the metallothionein-1 gene, could be added to linkage group VI (LG VI). Three microsatellite markers (one anonymous, one located within the PMP2 gene, and one located within the FABP6 gene) constitute a new linkage group (LG XI). We also measured the degree of dietary cholesterol-induced aorta atherosclerosis in the F2 animals. A significant cosegregation was found between the degree of aorta atherosclerosis and the allelic variation of the biochemical marker Est-2 on LG VI in male rabbits. This association was not found in female rabbits.
atherosclerosis; genetic map; quantitative trait locus; microsatellite
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INTRODUCTION
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THE RABBIT IS FREQUENTLY USED as an animal model for atherosclerosis and hypercholesterolemia (19, 27). To identify and localize the genetic factors that are involved in these diseases, a dense genetic map of the rabbit is needed. At present, the rabbit genetic map is still underdeveloped. Only 31 loci have been linked in 10 autosomal linkage groups (6). These loci have been detected by biochemical, immunological, or morphological methods. Although very useful in, for instance, homology studies, these markers are of limited value in gene mapping. Extending the rabbit genetic map with polymorphic markers at the DNA level would increase the value of such a map.
To date, primer pairs for 31 rabbit microsatellite markers have been published. The group of Hewitt and colleagues (24, 28; University of East Anglia, Norwich, UK) developed nine microsatellite markers from a genomic library screened with radioactive probes. Mougel et al. (20) published another nine microsatellites using the same technique for six of the microsatellites, whereas the remaining three were developed from extracted sequences of the EMBL nucleotide database. Van Lith and van Zutphen (31) screened the EMBL nucleotide database and found 157 rabbit nuclear gene microsatellites. Van Haeringen et al. (29) used the latter data and developed primers for 13 of these microsatellites. Linkage information is not available for any of the 31 microsatellite markers.
We here present some characteristics of another 23 rabbit nuclear gene microsatellites extracted from the EMBL nucleotide database. As these are located within sequences coding for functional genes and these genes are conserved across mammalian species, they can be considered as type I anchor loci (21). Nine of these 23 markers, together with 9 markers that were published earlier (20, 24, 28, 29), were found to be polymorphic between the AX/JU and IIIVO/JU inbred strains. These two strains differ in susceptibility to dietary cholesterol (17, 36). The AX/JU is a hyperresponder (high serum total cholesterol level after a cholesterol-rich diet) and develops in a relatively short time period dietary cholesterol-induced atherosclerosis. The IIIVO/JU is a hyporesponder (slight increase in serum total cholesterol level after a high-cholesterol diet) and develops no atherosclerosis. We analyzed the 18 polymorphic microsatellite markers together with the C (albino) locus and the previously described polymorphic biochemical markers (12) in an F2 intercross progeny derived from these strains. Furthermore, we measured the degree of atherosclerosis in the aorta of all F2 animals that had been fed a cholesterol-rich diet for 12 wk. Statistical analysis was performed to investigate a possible association between this trait and the markers that were genotyped.
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MATERIALS AND METHODS
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Microsatellite identification and primer design.
The EMBL nucleotide sequence database (version 32) was searched for all possible mono-, di-, tri-, and tetranucleotide rabbit microsatellites with a total length of at least 20 base pairs (31). Among those obtained, 23 microsatellite sequences were analyzed by PCR and electrophoresis. Based on the unique sequences flanking the microsatellites, PCR primers were designed using the computer program PRIMER (version 0.5; Whitehead Institute-Massachusetts Institute of Technology, Cambridge, MA; Lander ES, Green P, Abrahamson J, Barlow A, Daly MJ, Lincoln SE, and Newburg L, unpublished material, 1991). The primers were synthesized by Pharmacia Biotech Benelux (Roosendaal, The Netherlands) and tested at different annealing temperatures (55, 60, 65, and 70°C) and magnesium concentrations (1.0, 1.5, 2.0, 3.0, 4.0, and 5.0 mM) using genomic rabbit DNA. The primer sequences used for the amplification of these microsatellite loci are listed in Table 1. PCR was carried out in 10-µl reaction volumes containing 200 ng DNA, 720 nM forward primer, 720 nM reverse primer, 1x PCR buffer (HT Biotechnology, Cambridge, UK), 0.2 mM dNTP (HT Biotechnology), 15 mM MgCl2, and 0.3 U Supertaq (HT Biotechnology). After an initial 5 min denaturation at 94°C, 30 cycles were performed as follows: 30 s denaturation at 94°C, 1 min annealing at 55, 60, 65, or 70°C, and 2 min extension at 72°C. A final elongation was carried out for 10 min at 72°C. Products were separated in a 3% MS-8 Pronarose (Sphaero Q) gel and detected by ethidium bromide staining.
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Polymorphism and allele size determination.
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After optimizing the PCR conditions, we tested the microsatellite markers for polymorphisms using DNA samples from a test panel consisting of (partially) inbred strains (AX/JU, IIIVO/JU, OS/J, WH/J, and X/J), random-bred strains (New Zealand White, Californian, ELCO, and Watanabe), and wild rabbits (34). DNA was isolated as previously described in detail (29). PCR was performed under optimal conditions in 10-µl reaction volumes containing 20 ng DNA, 120 nM 32P-labeled forward primer, 120 nM reverse primer, 0.2 mM dNTP, and 1x PCR buffer with the appropriate concentration of MgCl2. The same PCR program was used as described above, with the optimal annealing temperature. Products were separated in a 6% denaturing acrylamide gel together with an allele sizing marker (10-base DNA ladder, Allele Sizing Set; Boehringer, Mannheim, Germany) and detected using autoradiography.
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F2 intercross progeny.
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The following two rabbit inbred strains are available at the Department of Laboratory Animal Science (Utrecht, The Netherlands): AX/JU, which is a dietary cholesterol-susceptible strain; and IIIVO/JU, which is a dietary cholesterol-resistant strain (17, 36). The two strains originate from the Jackson Laboratory colony, Bar Harbor, ME (5), and are distantly related to each other: the AX/JU strain originates from a chinchilla stock, and the IIIVO/JU strain originate from a New Zealand White breed. The history of the AX/JU and IIIVO/JU rabbit inbred strains has recently been described in detail (11). The two inbred strains are maintained by brother-sister mating, and the coefficient of inbreeding (F) > 0.98 for both strains. To produce F1 hybrids, IIIVO/JU females were mated with one AX/JU male. The F1 hybrids were intercrossed, producing 138 F2 progeny (61 males and 77 females). After weaning at the age of 10 wk, all F2 rabbits were fed a commercial diet (LKK-20; Hope Farms, Woerden, The Netherlands) and were housed individually. The chemical composition of the commercial rabbit diet has been described elsewhere (30). At 1216 wk of age, the commercial diet was replaced by the test diet, which contained 0.3 g of cholesterol (USP; Solvay Pharmaceuticals, Weesp, The Netherlands) per 100 g. The cholesterol-rich diet was fed during the test period, which lasted 12 wk. During the experiment, restricted amounts of diet (100 g·rabbit-1·day-1) were given each morning at 10.00 hours. On the day before killing the animals, any remaining food was removed at 16.00 hours. At the end of the test period, the fasted rabbits were anesthetized by an intravenous injection of Hypnorm (Janssen Pharmaceutica, Beerse, Belgium) sufficient to reach the surgical phase (
0.3 ml/rabbit). Subsequently, the animals were killed by cardiac exsanguination.
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Planimetry of the aorta.
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After killing, the aorta and the heart were removed en bloc and separated just above the aortic valve. The aorta was dissected from the arch of the ascending aorta to the ileac bifurcation of the abdominal aorta. The entire aorta was trimmed free of adventitial adipose tissue, opened longitudinally along the cranial wall with a fine scissors, pinned flat (using 0.2-mm diameter stainless steel pins) to a cork board, and longitudinally divided into three parts of about equal length (ascending aorta, thoracic aorta, and abdominal aorta). The ascending aorta contains the aortic arch. To enable a flat preparation of the aortic arch, the longitudinal cut was extended around the inner curve of the arch, and a separate cut was made along the outside curve of the arch. The aortic parts were then stretched between two glass plates and fixed in neutral aqueous phosphate-buffered formaldehyde solution. The fixed aortic parts were stained en face with a filtrated solution containing Sudan III and Sudan IV (1.0 g Sudan III and 0.5 g Sudan IV dissolved in 200 ml ethanol:acetone:H2O, 21:10:9, vol/vol/vol; Sigma Chemical, St. Louis, MO) to demonstrate areas of developed atheromatous plaques. The percentage surface area of the aortic luminal surface covered by sudanophilia, as shown by dark red regions corresponding to the uptake of Sudan III/IV by fat, was determined by computerized planimetry using a true-color image analyzer (VIDAS RT 2.5; Kontron, Munich, Germany). The analyzer was interfaced to a videocamera that was mounted on a dissecting microscope.
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Linkage analysis and statistical analysis.
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The F2 intercross animals have been genotyped for the C locus and the biochemical markers Mhr-1, Hp, Es-1, Est-2, Est-3, Est-4, and Est-6 as described elsewhere (12). By careful inspection of the liver esterase zymograms, we identified a new polymorphic esterase zone. We have provisionally given this new biochemical marker the symbol Est-X. DNA from the F2 intercross animals was isolated as described (29) and was used for the genetic analysis of the microsatellite markers. The segregation ratio for the individual markers was inspected by means of the
2 goodness-of-fit test. The genetic map distance for the markers was computed with the computer package Mapmaker/EXP 3.0 (14). For the establishment of linkage groups, we used a critical minimal LOD ("log of the odds ratio") score of 3.0. For calculation of map distances and estimating most likely gene orders, we used a critical LOD score of 0.05. Recombination frequencies were converted to map distances in centimorgans with the Kosambi function.
The Kolmogorov-Smirnov one-sample test was used to check normal distribution of the aorta atherosclerosis data. The significance of the differences between males and females was calculated with the unpaired Students t-test. The unpaired Students t-tests were performed with pooled (for equal variances) or separate (for unequal variances) variance estimates. The equality of variances was tested using an F test. quantitative trait loci (QTLs) affecting the degree of aorta atherosclerosis were mapped relatively to the linkage group (LG) VI markers with the use of the MapQTL version 3.0 software (35). Since the traits were normally distributed in both sexes, the interval mapping module was used (15). These results were expressed as LOD scores.
Comparison of the degree of aorta atherosclerosis of the F2 intercross animals after grouping by genotype has also been performed. For this purpose one biochemical marker (Est-2) was used. If the Est-2 locus and the degree of aorta atherosclerosis are segregating independently, then the values for aorta atherosclerosis will be equally distributed among the homozygote and heterozygote genotypes. The results for the degree of aorta atherosclerosis are presented as means ± SD. The Kolmogorov-Smirnov one-sample test was used to check normality of these data. All results within groups were found to be normally distributed. The significance of difference between the segregating genotype groups was calculated by two-way ANOVA with sex and genotype as factors. Homogeneity of variance was then tested using Bartletts test. The variances were similar. The probability of a type I error <0.05 was taken as criterion for significance. All statistical analyses were carried out according to Petrie and Watson (23) using an SPSS PC+ computer program.
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RESULTS
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Using the test panel of different rabbit breeds, we found that 10 of the 23 microsatellites were polymorphic microsatellites. Nine of these vary between AX/JU and IIIVO/JU (Table 2). In addition, nine previously described microsatellite markers [Ocwapg, Oc15lox, Ocp450II4 (29); Sat3, Sat4, Sat8, Sat13 (20); Sol33, Sol44 (28)] were also found to be polymorphic between AX/JU and IIIVO/JU.
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Table 2. Allele diversity of 10 microsatellites after screening in a testpanel consisting of 32 rabbits from different strains and breeds
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To conform rabbit microsatellite marker symbols with those of other mammalian species, we decided to follow similar rules. The basic rules of, e.g., rat gene nomenclature are recently described by Levan et al. (16). Markers that are not assigned to chromosomes are denoted D0Utr1, D0Utr2, etc. Markers of which the gene sequence is previously assigned to a chromosome [HBA to 6q12 (37), WAP to 10q16 (26), and the MHC complex to 12q11 (25)] are denoted D6Utr1, D10Utr1, and D12Utr1, respectively.
Although the AX/JU and IIIVO/JU are fully inbred strains and have been propagated for more than 20 consecutive generations by brother-sister matings, they still show intrastrain variation for several loci: D6Utr1 (AX/JU and IIIVO/JU), D0Utr2 (IIIVO/JU), D10Utr1 (IIIVO/JU), D0Utr3 (AX/JU), and D0Utr8 (IIIVO/JU). Although both AX/JU and IIIVO/JU strains were not uniform for D6Utr1 and IIIVO/JU not for D10Utr1, most of the parental animals used for the production of the F2 intercross were homozygous for D6Utr1 and D10Utr1. Therefore, these could be used for linkage analysis. We were unable to genotype the animals for D0Utr7. All 14 microsatellites used for genotyping the intercross inherited in a codominant, Mendelian fashion. Nearly all microsatellite markers segregated in the F2 as would be expected. Only the D10Utr1 marker showed a significant segregation distortion and therefore was not used for linkage analysis (Table 3). After analysis of the markers with the Mapmaker/EXP 3.0 computer program, 8 of the 13 microsatellite markers remained unlinked, suggesting that they are either on different chromosomes or are too far apart from one of the other markers. Sat13 was linked to the C locus and Est-5 (Fig. 1) and therefore could be assigned to rabbit chromosome 1 (12). The microsatellite in the metallothionein-1 (MT1) gene was linked to the esterase clusters of LG VI (Fig. 1). The three markers Sat3 (which is in the peripheral myelin protein 2 gene, PMP2), D0Utr6 (which is in the ileal lipid-binding protein gene, FABP6), and Sol33 are linked but could not be assigned to a chromosome or previously described linkage group.

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Fig. 1. Linkage maps of rabbit chromosome 1, linkage group (LG) VI, and a new linkage group (LG XI). For calculation of map distances, we used Mapmaker/Exp 3.0. The numbers to the left of the chromosome or linkage group indicate the distance between markers in cM.
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The degree of atherosclerosis was measured in the aortas of all F2 animals. Females, compared with males, developed a significantly higher degree of atherosclerosis in the aorta (Table 4). Therefore, for QTL analysis, we have split the data into a male and a female set. The values for the degree of atherosclerosis were found to be normally distributed both in males and females. We detected significant associations between markers on LG VI and the degree of aorta atherosclerosis in males. The LOD scores peaked in the vicinity of the Est-2 locus. In females we detected no significant associations with LG VI markers. Figure 2 shows the resulting plots of the LOD scores of different parts of the aorta and the total aorta against LG VI markers. Two-way analysis of variance applied to the genotypes of Est-2 confirmed that a genetic factor of LG VI is affecting the degree of aorta atherosclerosis in the rabbit (Table 5).

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Fig. 2. LOD score plots of the degree of atherosclerosis in the ascending aorta (A), thoracic aorta (B), abdominal aorta (C), and the total aorta (D) against LG VI. Squares, female data; circles, male data.
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DISCUSSION
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In this study we have used microsatellites located in or near genes. This might explain the low degree of polymorphism found. A polymorphism within an exon would be reflected in the protein of the gene, and therefore, with the exception of the D0Utr3 marker, no polymorphism was found when the microsatellite was located in an exon. Most polymorphisms were observed when the microsatellite was located at the 3' flank of the gene (Table 1).
All microsatellite markers tested on the F2 intercross, except D10Utr1, segregated according to the 1:2:1 distribution (Table 3). We have genotyped all available animals of the IIIVO/JU inbred colony for the D10Utr1 marker and found that most animals were heterozygous or homozygous for the allele found in the AX/JU strain (A allele), suggesting that there is a negative selection for the I allele. Thus, despite that the calculated F-value for the IIIVO/JU strain is high, there was intrastrain variation for the WAP (D10Utr1) locus. It is well known that the rabbit is a species with a severe degree of inbreeding depression (2). Thus it must be kept in mind that, because of this inbreeding depression, there is a simultaneous selection. For other rabbit milk protein loci, selection has also been described. For example, Bösze et al. (3) suggested selection for an allele at the rabbit kappa casein locus.
After linkage analysis, Sat13 was found to be linked to the linkage group on chromosome 1, and D0Utr5, the microsatellite in the metallothionein-1 (MT1), gene could be assigned to LG VI. When comparing this latter result with mouse, human, and rat, it is obvious that the MT1 homolog in these species is also located near the esterase clusters and haptoglobin (HP). Sol33, Sat3, (PMP2), and D0Utr6 (FABP6) form a new linkage group provisionally indicated as LG XI. The human homologs of the peripheral myelin protein 2 gene and the gene coding for ileal lipid-binding protein are located on 8q2122 and 5q2535, respectively. Comparative data suggest that in the rabbit these genes reside on chromosome 3 (10), with PMP2 on the q-arm and FABP6 on the p-arm.
As our two inbred strains differ in susceptibility to dietary cholesterol and the hyperresponder (AX/JU) also develops atherosclerosis in the aorta, the F2 population could be used in the search of QTLs, which are involved in dietary cholesterol-induced atherosclerosis. An important risk factor for atherosclerosis is a high serum total cholesterol level. However, not only the concentration of cholesterol in the blood is of importance, but also the lipoprotein profile. Numerous epidemiological and clinical trials have demonstrated a negative correlation between serum high-density lipoprotein (HDL) cholesterol levels and the development of atherosclerosis (7). We have shown previously that a genetic factor affecting basal serum HDL cholesterol level is located on LG VI, near Est-2 and Es-1 (32). Linkage homology has been shown for rabbit LG VI, rat chromosome 19, mouse chromosome 8, and human chromosome 16 (32). Interestingly, the gene for lecithin-cholesterol acyltransferase (LCAT) is located on human chromosome 16, rat chromosome 19, and mouse chromosome 8. This gene plays a major role in HDL cholesterol metabolism (8). Human chromosome 16 also contains the gene for cholesteryl ester transfer protein (CETP). This gene is also involved in HDL cholesterol metabolism (13). Meijer et al. (18) described that serum LCAT and CETP activities in AX/JU rabbits fed a cholesterol-rich diet were lower than in IIIVO/JU rabbits. Furthermore, it has been described that in humans, LCAT and CETP gene polymorphisms are associated with atherosclerosis (1, 9). Therefore, based on homology, one might speculate that in the rabbit the LCAT and CETP loci are also on LG VI, and thus one or both genes (or transcription factors in the vicinity of these genes) might be the responsible genetic factors for the strain difference in the degree of aorta atherosclerosis in cholesterol-fed rabbits.
The QTL for the degree of aorta atherosclerosis on LG VI is sex specific (Fig. 2 and Table 5). This is not unexpected since both laboratory animals and humans exhibit substantial sex-dependent differences in lipoprotein metabolism and susceptibility to atherosclerosis. The higher degree of atherosclerosis in F2 females compared with F2 males (Table 4) has also been observed in several atherosclerosis-susceptible mouse inbred strains. In these strains, the sex difference might be explained by the effect of testosterone on HDL cholesterol levels (22), although this seems to contradict to the human situation: within the human male population higher levels of testosterone are associated with higher HDL cholesterol levels and reduced risk of heart disease (4). However, for our F2 rabbits we feel that the higher circulating total cholesterol level in females when compared with males (33) is a major factor contributing to the sex difference in dietary cholesterol-induced aorta atherosclerosis. This hypothesis is consistent with the assumption that the severity of atherosclerosis is proportional to serum total cholesterol levels. It is not likely that the difference in serum HDL cholesterol concentration accounts for the higher degree of atherosclerosis in female when compared with male rabbits, since F2 males have lower circulating HDL cholesterol levels than F2 females (33).
In conclusion, this report describes the location of the first DNA markers in the rabbit genetic map. One microsatellite marker could be assigned to chromosome 1. One microsatellite marker could be added to LG VI, and three markers form a new linkage group (LG XI), which, on the basis of homology, is possibly on chromosome 3. Analysis of the genotypes of the F2 animals together with the data of the planimetry of the aortas revealed a significant cosegregation between the degree of aorta atherosclerosis and the allelic variation of Est-2 in male rabbits. This association was not found in female rabbits. When considering the homology of rabbit LG VI with human chromosome 16, rat chromosome 19, and mouse chromosome 8, LCAT and CETP (or their transcription factors) could be candidate loci involved in the differences in susceptibility of aorta atherosclerosis.

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Fig. 1. Linkage maps of rabbit chromosome 1, LG VI and a new linkage group (LG XI). For calculation of map distances the Mapmaker/Exp 3.0 was used. The numbers to the left of the chromosome or linkage group indicate the distance between markers in centiMorgans (cM).
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Fig. 2. LOD-score plots of the degree of atherosclerosis in the ascending aorta (A), thoracic aorta (B), abdominal aorta (C) and the total aorta (D) against Linkage group VI. ( = female data, = male data).
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FOOTNOTES
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Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).
Address for reprint requests and other correspondence and current address of R. Korstanje: The Jackson Laboratory, 600 Main St., Bar Harbor, ME 04609 (E-mail: rkorstan{at}jax.org).
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