Quantitative trait loci on chromosome 1 for cataract and AMD-like retinopathy in senescence-accelerated OXYS rats

Cataract and retinopathy incidence in the parental population and F1, F2 hybrid rats

Manifestations of cataract and retinopathy are absent in the eyes of OXYS rats at the age of 20 days. The first AMD-like alterations in retina and signs of cataract develop in 20-22% of OXYS rats when they reach the age of 1.5 months. In recent years, the morbidity of both cataract and retinopathy has reached 100% by the age of 3-4 months. Alterations of retina are manifested as distorted reflectance of the eye ground, swelling of the retina, the emergence of distinct foci of ischemia, and the signs of atrophy of choriocapillaris and retinal pigmented epithelium (RPE). These correspond to the first stage of AMD [18, 39]. The majority of animals at this age are also characterized by lens alterations corresponding to stage 1 of cataract and exhibit zonal cortical or nuclear opacities, but 25-40% of OXYS rats already have a second stage cataract [28]. When the generation of hybrids was started, the morbidity of OXYS rats was somewhat different. The results of ophthalmoscopic examination of the parental animals are shown in Table 1. The incidence of cataracts in 3-month-old OXYS rats was independent of sex according to the data, while the incidence of retinopathy was ~ 1.3 times higher in females. This is consistent with published data on the development of AMD in humans.

The results of the examination of F1 and F2 hybrids are presented in Tables 1 and 2. In F1 hybrids significant sex differences were also observed, the incidence of cataracts was 3.1, and retinopathy 3.7 times higher in females than in males. The influence of gender on the incidence of ocular pathologies was confirmed by regression analysis (for retinopathy: R = 0.546, R² = 0.300, adjusted R² = 0.281; F (1.37) = 15.842, p <0.00031; for cataract: R = 0.587, R² = 0.345, F (1.37) = 19.5, p <0.00009). There were no significant differences in the disease severity between the groups F1 (female OXYS × male WAG) and F1 (female WAG × male OXYS) (Table 2). Therefore, the data from the studies of reciprocal F1 hybrids were combined (Table 1). After that, the sex differences became even more significant: F(1.42) = 24.2, p = 0.00001 (for cataract) and F(1.42) = 22.5, p = 0.00002 (for retinopathy). In the second-generation (F2) hybrid population, the morbidity of both cataract and retinopathy significantly increased compared to the first generation (F1) only in male rats, and there were no sex differences (Table 2).

QTL analysis

Identification of genetic loci responsible for the traits of interest was performed on hybrid F2 population at age 3-4 months. The population of 77 F2 hybrid rats showed two loci with highly significant associations of markers on chromosome 1 with the development of cataract and retinopathy. Figure 1 shows the plots of distribution of the probability of linkage between the markers analyzed and hypothetical genes controlling a trait. The locus on chromosome 1 controlling cataract development was located in the region of markers D1Rat219 and D1Rat81 (LOD score 3.07); the loci controlling retinopathy were located in the regions of markers D1Rat30 and D1Rat219 (LOD score 4.89), as well as D1Rat219 and D1Rat81 (LOD score 7.79).

Figure 1. Plots of the distribution of trait linkage probability in the population of F2 rats (bred by reciprocal cross of OXYS and WAG) at age 3-4 months. The linkage of: (panel A) retinopathy; (panel B) cataract with markers on chromosome 1 was analyzed. Dotted vertical line at LOD = 2.0 is the threshold for suggestive statistical significance. The OXYS regions introgressed into WAG rats in the construction of congenic strains are shown as inserts to the left of the linkage map.

Generation of congenic rats

The construction of a congenic strain is a standard procedure of experimental mammalian genetics [4, 12, 37] originating from the work of G.D. Snell [33] on histocompatibility loci. Figure 2 outlines the procedure for moving a polymorphic marker gene from one inbred (donor) strain to another inbred (recipient) strain. In our study congenic strains were produced in accordance with the traditional approach: a QTL allele from the OXYS strain was introgressed into the WAG strain, which showed no signs of cataract and retinopathy development. The transfer of the OXYS donor region was accomplished by first producing F1 offspring by the reciprocal crosses of donor OXYS strain with recipient WAG strain (female WAG×male OXYS and female OXYS ×male WAG) and then backcrossing F1 rats with rats of a recipient WAG strain to obtain BC1 progeny. In Figure 2, the donor at marker locus M has genotype M1M1, and the recipient is M2M2. Tail tip DNA was isolated by the standard proteinase K digestion method and offspring rats were genotyped to identify animals heterozygous for a desired chromo-some 1 segment (5 markers were used: D1Rat30, D1Rat54, D1Rat219, D1Rat117, D1Rat76, and D1Rat81) and homozygous for the highest amount of recipient alleles at markers outside the chromosomal region of interest. These animals (‘carriers’) were backcrossed with new WAG rats to generate BC2 progeny for genotyping. Thus, 8 backcross generations characterized by subsequent genotyping were produced, as shown in Figure 2. This was done to ensure that nearly 99% of the donor's OXYS genetic background had been replaced by that of the recipient WAG [26]. Upon completion of these 8 backcrosses, brother-sister mating of two heterozygotes created the desired chromosomal regions homozygous for the donor's alleles and yielded offspring in the ratio M1:2M1M2:1M2M2. Two homozygous offspring M1M1 were bred to fix the M1 donor marker allele (along with a cosegregating locus of interest) in the recipient background. The rats of the two congenic strains were designated formally as WAG /OXYS-1.1 and WAG /OXYS-1.2.

Figure 2. The breeding scheme for the construction of congenic strains. Selection is made at microsatellite marker locus M through a series of backcrosses such that the M₁ allele of a donor (OXYS) replaces the M₂ allele of a recipient (WAG). Rats are genotyped at M by PCR reaction using DNA obtained from tail tips. The genome of the donor strain is shown as an open symbol, and the genome of the recipient strain as a solid symbol. Increasing shades of gray from light to dark represent an increase in the percentage of genetic background of the recipient that occurs with each backcross (BC). The scheme is based on [26].

The aim of the subsequent experiments was to assess the effect of these presumptive QTLs on disease manifestation in the congenic rat strains. Congenic rats serve as “proof of concept” that a QTL, when placed in another genetic background, has a meaningful effect in vivo on a particular phenotype [4].

Characteristics of congenic rats

In the process of constructing congenic strains, the QTL allele from the OXYS strain was introgressed into the WAG strain, which showed no signs of cataract and retinopathy development. Two congenic regions of chromosome 1 are shown in Figure 1 as inserts to the left of the linkage map: loci marked by D1Rat30 and D1Rat219 (the region named QTL1: 100,6–188,0 Mb) and by D1Rat219 and D1Rat81 (region, named QTL2: 188,0–250,4 Mb). Figure 3 shows data on cataract and retinopathy morbidity and sickness rate for each congenic strain. The expected results were that the WAG/OXYS-1.1 congenic strain would develop retinopathy, and the WAG/OXYS-1.2 strain would develop both early cataract and retinopathy.

Figure 3. Cataract and retinopathy incidence in rats of WAG/OXYS-1.1 and WAG/OXYS-1.2 congenic strains at the ages of 1.5-2 and 3-4 months. Data are presented as a distribution of animal eyes with the stages of cataract and retinopathy (colors labeled with 0 through 2 correspond to a stage of cataract or retinopathy).

At the age of 1.5-2 months, in 54% of the eyes of WAG/OXYS-1.1 congenic animals, we found cortical and posterior subcapsular opacities corresponding to the first stage of cataract. For animals of the WAG/OXYS-1.2 strain the percentage was 76% of the eyes for the stage 1 cataract and 9% of the eyes for the second-stage cataract. The percentage of afflicted animals increased with age. In the group aged 3-4 months 93% of eyes were affected by the first-stage cataract in WAG/OXYS-1.1, but there were no cases of stage 2 cataract. The incidence of cataract had reached 100% in animals of WAG/OXYS -1.2 by age 3-4 months, and in 34% of the eyes the pathological changes matched the 2nd stage of the disease.

The cataract developed against the background of progressive retinopathy in congenic animals like in OXYS rats. At the age of 1.5-2 months, the signs of stage 1 retinopathy were found in 11% of the eyes of WAG/OXYS-1.1 rats. At the age of 3 months the retinopathy incidence in this strain was 55%. Interestingly, these values were approximately 2-fold lower than those in OXYS rats. In WAG/OXYS-1.2 animals at age 1.5-2 months, stage 1 retinopathy was observed in 41% of the eyes, but at 3-4 months 97% of the eyes were affected. Nonetheless, the animals of both congenic strains contained no cases of stage 2 retinopathy.

Experimental animals and ophthalmoscopy examination

We used OXYS rats, rats of WAG (Wistar Albino Glaxo) strain with no signs of age-related eye diseases, F1 and F2 hybrids obtained by the reciprocal crosses (female WAG×male OXYS and female OXYS ×male WAG), and rats of backcross and congenic populations. All animals were born and reared at the Center for Genetic Resources of Laboratory Animals at the Institute of Cytology and Genetics (ICG), Siberian Branch of the Russian Academy of Sciences (SB RAS, Novosibirsk, Russia). All experiments in this study were approved by the Institutional Review Board and performed in accordance with Animal Care Regulations of ICG and with the international norms for studies with laboratory animals. At the age of 4 weeks, the pups were taken away from their mothers and housed in groups of five animals per cage (57×36×20 cm) and kept under standard laboratory conditions (22±2°C ambient temperature, 60% relative humidity, and natural light), provided with a standard rodent feed, PK-120-1, Ltd. (Laboratorsnab, Russia), and given water ad libitum.

Ophthalmoscopic examination was carried out using Beta direct ophthalmoscope (Germany) equipped with a slit lamp, after dilatation with 1% tropicamide. Ophthalmoscopic examination of rats of the parental strains (OXYS and WAG), second-generation (F2) hybrids and congenic animals was performed at ages 1.5-2 and 3-4 months. All animal procedures were in compliance with the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research as well as the European Communities Council Directive No. 86/609/EES. Assessment of stages of cataract and retinopathy was carried out according to the Age-Related Eye Disease Study (AREDS) grade protocol (http://eyephoto.ophth.wisc.edu). The following grades of cataract were used: 0 – transparent lens; 1 – very light cortical or nuclear opacity in the lens (corresponding to the decimal scale of the standard 1-4); 2 – opacity zones (corresponding to the decimal scale of the standard 5-8); 3 – intensive cortical or nuclear opacity of the lens (corresponding to the decimal scale of the standard 9-10). The degree of retinopathy was estimated as follows: 0 – arbitrary unit corresponding to healthy retina; 1 – appearance of drusen and other pathological changes in the retinal pigmented epithelium and partial atrophy of the choroid capillary layer; 2 – exudative detachment of RPE and of retinal neuroepithelium, with further choroid capillary layer atrophy.

DNA extraction

DNA was extracted from tail tips using proteinase K and phenol extraction using standard methods. DNA was then reprecipitated and dissolved in deionized water, and DNA concentration was measured using Eppendorf Biophotometer (Germany).

Microsatellite markers and polymerase chain reaction (PCR) on rat genomic DNA

Microsatellite markers were selected from the http://www-genome.wi.mit.edu and http://www.well.ox.ac.uk databases. The relative positions of markers on chromosomes, expressed in millions of nucleotides (megabases, Mb), were determined using the rat genome sequence at http://www.ensembl.org/. Twenty six microsatellite markers located on the rat chromosome 1 were tested. Of these only 13 showed interstrain polymorphism and intrastrain homogeneity during the testing of genomic DNA from OXYS and WAG rats. These markers were used in analyses of DNA from the population of F2 hybrids at 3-4 months of age (males and females). The ratio 1:2:1 as predicted by theory held for only 5 of 13 selected markers when we calculated the three marker classes ratio (homozygote by the first allelic variant, homozygote by the second allelic variant, and heterozygote), as estimated by χ² criterion. These 5 markers were used in the following QTL-analysis: D1Rat196 (55.1 Mb), D1Rat224 (90.3 Mb), D1Rat30 (100.6 Mb), D1Rat219 (188.0 Mb), and D1Rat81 (250.4 Mb). The three markers that are located within the chromosomal regions of interest (D1Rat54, D1Rat117, and D1Rat76) were used to select the most appropriate animals in the backcross population for producing the next generation. The primer sequences for the markers used are given in Table 3.

PCR was performed in 1 × buffer (67 mM tris-HCl (pH 8.9), 16 mM (NH₄)₂SO₄, 1.5 mM MgCl₂, 0.01% Tween 20, and 10 mM β-mercaptoethanol) containing each of the four dNTP at 200 μM, each primer at 3 μM, with the addition of 50-100 ng of DNA, and 1 activity unit of Taq I DNA polymerase (Institute of Cytology and Genetics, SB RAS, Novosibirsk, Russia) in 25 μl reaction volume. Cycling conditions were as follows: initial denaturation at 95°C for 5 min, followed by 38 amplification cycles of denaturation at 95°C for 15 sec, primer annealing for 20 sec, and elongation at 72°C for 20 sec. The final elongation was 72°C for 5 min. We conducted a preliminary experiment to define optimal annealing temperature for each primer pair (shown in Table 3).

Electrophoretic analysis of PCR fragments

PCR fragments were analyzed by electrophoresis in 6% polyacrylamide gels. Electrophoresis was performed in tris-borate buffer (0,5×TBE) at a field strength of 10 V/cm. Separated PCR fragments were visualized by EtBr staining.

Statistical and linkage analysis

The data were analyzed using repeated measures ANOVA with the statistical package Statistica 6.0 (StatSoft, Tulsa, USA). Two-way ANOVA was used to assess gender differences in genotypes (sex × genotype). A Newman-Keuls post hoc test was applied to significant main effects and interactions in order to estimate the differences between particular sets of means. One-way ANOVA was used for individual group comparisons. Results were considered statistically significant if p value was less than 0.05.

Linkage of markers with hypothetical genes controlling the trait of interest was assessed by calculating the plausibility criterion (odds ratio) for and against linkage. The plausibility criterion for QTL is the LOD score (logarithm of odds). Linkage was considered significant when the LOD score was greater than the threshold value of 3.0. Linkage analysis was performed using the MAP MAKER/EXP 3.0 and MAPMAKER/QTL 1.1 programs (Whitehead Institute, Cambridge, MA) [15, 16].