A Strong and Highly Significant QTL on Chromosome 6 that Protects the Mouse from Age-Related Retinal Degeneration

Abstract

purpose. BALB/cByJ (C) albino mice have significantly more retinal degeneration as they age than C57BL/6J-c^2J (B6) albinos. To discover the genetic loci that influence age-related retinal degeneration (ARD), a quantitative genetics study was performed with 8-month-old progeny from an intercross between these two strains.

methods. The thickness of the outer nuclear layer of the retina was used as the quantitative trait. A genome-wide scan was performed with 86 genetic markers at an average distance of 15.7 cM. Map Manager QTX was used to analyze the data.

results. Three highly significant quantitative trait loci (QTLs) were detected on mouse chromosomes (Chrs) 6, 10, and 16. The B6 alleles were protective against ARD in the first two, and the C allele was protective in the third. Several suggestive, weak QTLs were also found, along with a gender-related effect. The strongest and most highly significant QTL on Chr 6 accounted for 30% of the total genetic effect with a LOD score of 13.5. The RPE65/MET450 variant of major influence on constant light-induced retinal degeneration (LRD) in a previous study of these same two mouse strains had no influence on ARD, and only some of the weak, suggestive QTLs influencing ARD were also observed in LRD.

conclusions. Because none of the ARD QTLs was homologous to human chromosomal loci so far implicated in age-related macular degeneration, each represents a new candidate gene for potential study. The gene represented by the Chr 6 QTL is of particular interest because it has broad influence, very high significance, and a B6 allele that protects against ARD.

Age-related macular degeneration (AMD) is the most common cause of severe, irreversible vision loss in developed nations, ^{1 2 3 4 5} but little conclusive information is known about its etiology. Many studies have attempted to determine environmental risk factors that may be associated with AMD, but only smoking has been consistently demonstrated to be one of them (for reviews, see ^{6 7 8 9} ). In contrast, twin studies and population-based familial aggregate studies have made it clear that genes play a significant role in AMD. ^{10 11 12 13} It is also clear that AMD is a complex genetic disorder, ^{14 15} one that first appears most commonly in elderly individuals, typically in those older than 50 years. Because of the age of onset, informative family pedigrees of the size needed to identify genetic loci are difficult to find. Only one such locus (with a LOD score of 3.0) has been reported. ¹⁶ Identifying AMD genetic loci with family-based aggregate studies is difficult for the same reason and because the phenotype of AMD is heterogeneous. Is it one disease or a group of diseases? Are different phenotypes caused by the same or various genetic factors? To our knowledge, only one study of this type has been reported. In the latest refinement of this study, several loci were found in a very large cohort with LOD scores ranging from 2.0 to 3.16. ¹⁵  

Therefore, when we observed that two albino mouse strains undergo significantly different rates of retinal degeneration as they age, ¹⁷ we decided to perform a quantitative genetic study to find the chromosomal loci of the mouse genes responsible for the difference. This would be the first step toward finding the genes themselves—particularly the protective alleles of those genes—that influence age-related retinal degeneration. Compared with studies in humans, experiments with mice are much simpler to perform and are statistically more powerful. There are only two alleles to consider, one from each of the inbred strains. There is no problem with variation in phenotype, because measurement of the thickness of the outer nuclear layer (ONL) of the retina serves as a quantitative trait. There is no problem with confounding environmental influences because all the mice are exposed to the same conditions in the vivarium, and there is no problem recruiting subjects. 

Genes found to influence age-related retinal degeneration in the mouse would be excellent candidates for study in human AMD (even though the mouse does not have a macula or a fovea), because mouse and human genes involved in vision are often similar in effect. This is exemplified by the fact that mutations in several mouse vision genes such as Pdeb, Prph2, and Nr2e3 cause the same type of disease in mice that mutations in human orthologous genes (PDE6B, RDS-peripherin, and NR2E3) cause in humans. ^{18 19 20 21 22 23 24 25 26 27 28}  

In this study, with a large F1 intercross between the mouse strains BALB/cByJ (C) and C57BL/6J-c^2J (B6), we identified a number of quantitative trait loci (QTLs) containing genes that influence age-related retinal degeneration. Among them were three strong and highly significant QTLs and several weaker QTLs. Although most of the QTLs reflected B6 alleles that were protective, a few were the opposite; C alleles were protective. In addition, although the net relationship between B6, F1, and BALB/cByJ age-related, retinal degeneration control animals was dominant for B6, QTLs with additive and recessive relationships also were found once the loci were teased apart. Although the C57BL/6J-c^2J albino isogenic to C57BL/6J was a good choice to cross with the albino BALB/cByJ, it introduced potential epistatic confounds. The c allele (mutant tyrosinase gene) may have masked retinal degeneration gene alleles that would have been expressed on a pigmented C57BL background or exposed alleles that would not have been expressed on that background. Last, because we used these same two strains of mice in a previous quantitative genetic study of light-induced retinal damage, ¹⁷ we were able to compare the QTLs between the two studies to determine what influence the genes that modify light-induced retinal degeneration have on age-related retinal degeneration. 

Materials and Methods

Mice

BALB/cByJ and C57BL/6J-c^2J albino mice were originally purchased from the Jackson Laboratories (Bar Harbor, ME) although some were maintained through many generations in our vivarium before study. C57BL/6J-c^2J mice are derived from a C57BL/6J strain that underwent a mutation that inactivated the tyrosinase gene (c) to make it albino. Therefore, the strain is isogenic with C57BL/6J. By convention, the abbreviation for C57BL/6J mice is “B6” or “B” and for BALB/cByJ mice is “C” or “CBy.” All mice were kept under a 12-hour light–dark cyclic light cycle with an in-cage illuminance of 2 to 7 ft-c. The temperature of the vivarium was maintained between 18°C and 20°C. Cages were kept on four shelves of free-standing, five-shelf racks (never on the top shelf). Each week, the cages were rotated by shelf, by side of the rack (left or right), and by position on the shelf (seven positions from front to back). Mice were maintained on a low-fat diet (15001 Rodent Laboratory Chow; Newco Distributors, Rancho Cucamonga, CA) with chow and water available ad libitum. 

For the quantitative genetic study, a nonreciprocal (BALB/cByJ x C57BL/6J-c^2J)F2 cross was made, and 268 F2 progeny were aged to 8 months along with 30 BALB/cByJ, 23 C57BL/6J-c^2J and 50 F1 control mice. Because the mothers of all the F1s were BALB/cByJ, all F1 and F2 mice had the CBy mitochondrial genome and all F1 and F2 males had the B6 Y chromosome. 

Quantitative Traits

After the mice were aged to 8 months, eyes were enucleated immediately after death, fixed in a mixture of 2% formaldehyde and 2.5% glutaraldehyde in phosphate buffer, embedded in an Epon-Araldite mixture, and bisected along the vertical meridian through the optic nerve head. A single 1-μm section was taken from the cut surface of one of the half-orbs from each mouse and stained with toluidine blue, as described previously. ²⁹ On this section, measurements of the thickness of the ONL were made. Three measurements, each spaced 50 μm apart, were taken at nine 0.25-mm intervals, both in the superior and inferior hemispheres starting from the optic nerve head. The means of 54 measurements from the entire retinal sections were used to score the mice for the quantitative trait. For the purpose of comparison with results in a previous light-damage study, ¹⁷ the means of 12 measurements from the posterior retina in the superior hemisphere were used for the quantitative trait (Fig. 4 , areas marked 2–5). All procedures involving the mice adhered to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and the guidelines of the Loyola Marymount University Committee on Animal Research. 

Genotyping

Genotyping services were provided by the Center for Inherited Disease Research (CIDR; The Johns Hopkins University, Baltimore, MD). For each chromosome, the most proximal marker genotyped was within 15 cM of the centromere, internal markers were no more than 30 cM apart, and the most distal markers were within 15 cM of the telomere. The average spacing was 15.7 cM. The exceptions were Chr 1 where the two most distal markers were 45 cM apart, Chr 8 where the two most proximal markers were 39 cM apart, and Chrs 2, 5, 11, 12, and X where the most proximal markers were 48, 20, 17, 16, and 17 cM from the centromere, respectively. A list of markers used in crosses between C57BL and BALB/cByJ mice is available on the CIDR Web site (http://www.cidr.jhmi.edu/). A few additional dinucleotide repeat markers within QTLs were analyzed in our laboratory. These were amplified by standard PCR methods and electrophoresed in 4% agarose gels for allele determination by size. All map positions were based on the Encyclopedia of the Mouse Genome from Jackson Laboratories Mouse Genome Informatics (MGI; http://www.informatics.jax.org/; provided in the public domain by Jackson Laboratories, Bar Harbor, ME). 

Mouse Genomic DNAs

Genomic DNAs were isolated from liver tissue with a kit (Puregene; Gentra Systems, Minneapolis, MN). 

Data Analysis

Genotypes versus quantitative traits were analyzed with the Map Manager QTb17X program ³⁰ (http://mapmgr.roswellpark.org/mapmgr.html/; provided in the public domain by Rosewell Park Cancer Institute, Buffalo, NY). With this program, a likelihood ratio statistic (LRS) was calculated for each of the 86 marker genotypes, with a probability inclusion level for further study of 0.05. Thus, any single-point LRS for a marker that had only a 5% probability or less of occurring by chance in this set of data was included for further study. Of these markers, the one with the highest LRS was studied by interval mapping of all the markers on its chromosome. The marker at the peak of this QTL was put into the background for the next evaluation. Then peak markers from both the first and second interval maps were put into the background for the next determination, and peak markers from the first, second, and third QTLs were put into the background for the next determination. However, after the three strong and highly significant QTL markers were placed in background, the remaining LRSs were all in the suggestive category and very close to one another. Therefore, each of these was evaluated only with the three markers from the highly significant QTLs. To determine significance levels for this genome-wide screen, a test of 1000 permutations of all marker genotypes together was performed. For the quantitative trait of the entire retinal section (the mean of 54 measurements), P < 0.001 was 22.9 (highly significant [HS]); P < 0.05 was 16.0 (significant [S]) and P < 0.67 was 9.1 (suggestive or sugg). A highly significant LRS of 22.9 or more had a 99.9% probability or more of being real and would only occur by chance in 1 of 1000 genome scans such as this one. For the quantitative trait of the posterior superior section (Fig. 4 , positions 2–5) HS was 24.3, S was 15.2, and sugg was 9.2. The LRS was converted to an LOD score by dividing by 4.6 (2 × the natural log of 10). 

Results

Age-Related Retinal Degeneration QTLs

We used the thickness of the retinal ONL consisting of the intercalated and packed photoreceptor nuclei as the quantitative trait reflecting retinal degeneration. A thinner ONL corresponds to fewer photoreceptor nuclei and a greater loss of photoreceptor cells. To determine the best age at which to measure the quantitative trait, we measured the ONL in control BALB/c and B6 mice at ages 6, 8, 10, and 12 months (Fig. 1) . The age of 8 months was selected for study because this was the earliest age showing a significant difference. With evaluation of additional 8-month-old control animals, it was observed that the average ONL thickness was not significantly different between B6 and F1 retinas, whereas both were significantly thicker than those of BALB/c (Table 1) . This suggested a net autosomal dominant relationship between the aging retinal degeneration-influencing alleles of the two strains. For the study, F1 mice from the B6 and C strains were intercrossed to produce 268 F2 progeny (536 meioses) that were aged to 8 months in dim cyclic light. 

Using the Map Manager QT17X program ³⁰ to analyze the ONL thickness data of the F2 progeny versus the genotype data from a genome-wide scan performed with dinucleotide repeat markers, we found three highly significant and strong QTLs and five weaker QTLs. Table 2 shows that based on 23 B6, 30 C, and 50 F1 control ONL retinal measurements (103 control animals), MAP Manager QTX calculated that 69% of the observed age-related effect was due to genetics. Of that 69%, 21%, or 30% (21/69) of the total genetic effect was due to a gene acting in a QTL on mid-Chr 6 (Figs. 2a 2b) . The LOD score for this QTL was 13.5 and the B6 allele was dominant. QTLs on Chrs 10 and 16 were each 13% of the total genetic effect (Figs. 2c 2d 2e 2f ; Table 2 ) with LOD scores of 6.4 and 7.1, respectively. The B6 allele for the Chr 10 QTL was additive with the C allele; the B6 allele for the Chr 16 QTL was recessive/additive—that is, the Chr 16 B6 allele influenced the C allele but substantially less than 50%, or in other words, the B6 allele was recessive but not completely recessive. Purely additive alleles would have influence on one another equal to that of those acting in the Chr 10 QTL. Each of these first three QTLs was highly significant—that is, the probability that they would occur by chance in a genome-wide screen of the size and complexity of this study was less than 0.001). However, although the QTLs of Chrs 6 and 10 reflected B6 protective gene alleles, the Chr 16 QTL reflected a B6 allele that did not protect the retina from age-related retinal degeneration. In this case, it was the C allele that was protective. 

The five weaker QTLs were all suggestive, with LOD scores ranging from 2.1 to 2.6, and equivalent to only 4% of the genetic effect each. In four of these QTLs, the B6 alleles were protective; in the fifth on Chr 12, the C allele was protective (Table 2) . A suggestive QTL (P < 0.67) means that for every three genome scan studies similar to this one, two of these (suggestive) associations will occur by chance. Therefore, independent confirmation is needed to verify these suggestive QTLs. 

To determine whether any genes were acting together to influence age-related retinal degeneration in a significant, synergistic way, we used the interaction function of Map Manager QTX. For an intercross, this function tests every marker as an additive and dominant allele against every other marker as additive and dominant (four interactions per pair of markers). The interaction LRS (IX) needed for significance is approximately 20 (LOD score of 4.35) for an intercross. ³¹ When this function was performed with an exclusion probability of 10⁻⁵ or less (as the program recommends), only one interaction was found, and it was of questionable significance. The interaction involved the markers D9Mit355 (mid distal Chr 9) and D18Mit208 (middle Chr 18). The IX was 19.9 (LOD score = 4.33). Otherwise, there were no other significant interactive gene effects, as represented by IX scores of less than 20 for all marker (chromosomal loci) interactions of the genome-wide scan. 

The X and Y Chromosomes

There was a gender-related effect on age-related retinal degeneration. The average ONL thickness in 25 F1 control males was significantly greater than that in 25 F1 females (49.42 μm vs. 45.17 μm; unpaired Student’s t-test P = 3.21 × 10⁻¹⁰), and the average ONL thickness in 148 F2 males was significantly greater than that in 120 F2 females (45.17 μm vs. 43.41 μm; P = 0.0016). Because the test intercross was nonreciprocal, all the mothers of the F1s were BALB/cByJ mice. For this reason, and for the reason that the Map Manager QT program does not distinguish between hemizygous male and homozygous female genotypes for the interval mapping function in intercrosses, we evaluated the influence of the loci on the X chromosome by other means. 

For each of the four X chromosome markers, we calculated the average ONL thickness for F2 males hemizygous for the C allele, F2 males hemizygous for the B6 allele (B), F2 females homozygous for the C allele, and heterozygous F2 females (because of the breeding plan, there were no F2 females homozygous for X chromosome B6 alleles). Figure 3a shows the average ONLs of the F2 progeny with the various genotypes. The males consistently had ONLs thicker than those in females, and males hemizygous for C consistently had ONLs thicker than did males hemizygous for B. To evaluate the significance of this, we performed t-tests comparing average ONL thickness in the two male and two female F2 genotypes at each marker. 

Figure 3b shows no significant difference between the females homozygous for C and those heterozygous at any of the four markers. Therefore, we compared all females with males hemizygous for B and with males hemizygous for C, because F2 males on average had thicker ONLs than did F2 females. Males hemizygous for B was barely (at DXMit68) or nearly significantly different from F2 females (at the other three markers) suggesting that the B6 Y chromosome provides a small measure of protection against age-related retinal degeneration. This was supported by the fact that 12 male 8-month-old B6 control mice had on average significantly thicker ONLs than did 11 B6 8-month-old female control animals (47.3 μm vs. 45.02 μm; P = 0.029). There was no significant difference in ONL thickness between 12 male and 18 female 8-month BALB/c control animals (38.93 μm vs. 39.30 μm; P = 0.463). 

The males hemizygous for C were significantly different from all females, with a peak at the marker DXMit216. They were also significantly different from the males hemizygous for B, but only at DXMit216 (Fig. 3b) . This suggests that the BALB/c X chromosome carries a gene near the DXMit216 locus that contributes to protection against age-related retinal degeneration, but only when the B6 Y chromosome is present. If the BALB/c X chromosome locus at DXMit216 could provide protection without the B6 Y chromosome, the female F2 mice homozygous for the X chromosome C allele at that locus would show average ONLs similar to those in males hemizygous for the same allele. As shown in Figure 3 , this was not the case. 

Discussion

In a genetic study of the BALB/c and C57BL/6J-c^2J albino strains of mice, we have identified three highly significant and strong QTLs that influence age-related retinal degeneration. Two of these loci on Chrs 6 and 10 represent genes with B6 alleles that are protective, whereas the third on Chr 16 has a gene with a C allele that is protective. 

Because age has been a consideration in retinal light-induced damage in animal models, ^{32 33 34} we compared the results of the present study with results from a previous light-damage study in these same two mouse strains. The quantitative trait used in the earlier light-damage study ¹⁷ was based on ONL thickness measurements taken from the more light-sensitive superior, posterior retinal hemisphere (Fig. 4 , positions 2–5). However, in the current aging study, we measured the ONL in 18 positions across the entire length of the vertical retinal section. For the purpose of comparison, we recalculated the ONL thickness of the 268 F2 progeny of this aging study in only positions 2 to 5 in the superior posterior retina. We then calculated QTLs with this quantitative trait (Table 3) . 

The same three QTLs that were strong and highly significant in the entire retinal section were strong and highly significant in just the superior, posterior hemisphere. Among the weaker, suggestive QTLs, the Chr 8 locus was no longer significant when calculations were based on just the superior, posterior hemisphere, but a locus at mid-Chr 9 became suggestive. An additional locus on distal Chr 14 also became tentatively suggestive. It was just a bit below the LRS cutoff for a suggestive QTL (9.1 vs. 9.2). The other four pairs of loci present when either form of the quantitative trait was used (including the Chr 12 QTL), were similar in strength (Tables 2 3) . 

The three strong age-related retinal degeneration QTLs had either no influence or very little influence on the genetics of constant bright light-induced retinal degeneration. The QTL on Chr 6 could not be assessed for its influence on light-induced retinal damage because of the possibility that the B6 allele of the gene in this QTL was dominant in the previous study as it was in the current age-related retinal degeneration study. A QTL with a dominant B6 gene allele would have been hidden in the genetic backcross of the light-damage study. Nevertheless, age-related retinal degeneration is significantly influenced by at least two genes that have little or no influence on the type of constant bright-light–induced retinal degeneration we studied previously. ¹⁷ In addition, the RPE65-MET/LEU450 variant on distal Chr 3, which accounted for nearly 50% of the genetic response influencing light-induced retinal degeneration, had no influence on age-related retinal degeneration. Thus, substantial genetic portions of these two causes of retinal degeneration are distinct from each another. 

This is not to say that there is no overlap. There were six suggestive QTLs in the aging study that were calculated using the average ONL thickness of the posterior superior retina. Three of these suggestive QTLs were in common with QTLs from the light-damage study. The two QTLs on Chrs 9 and 12 that represented C alleles that protect the retina from light-induced retinal damage also protect the retina from age-related retinal degeneration. The one QTL on Chr 14 that was protective against light for B6 was also protective against aging. They were also similar in strength: each of the three QTLs accounted for only a few percentage points of the genetic effect in both studies. The presence of these three QTLs in two separate studies and with similar (but small) effects in each study verifies them and shows that at least a small part of age-related retinal degeneration may be influenced by the same genes that influence light-induced retinal degeneration. 

In humans, the question of sunlight exposure as an environmental factor in AMD has been investigated extensively, but no clear answer has emerged. ^{6 7 8 9 35 36 37 38 39} The genetic factors that influenced light-induced retinal damage in our studies accounted for a small percentage of the genetic effect in age-related retinal degeneration (not including the unknown influence on light-induced damage of the B6 dominant Chr 6 QTL). Perhaps, in the same way, sunlight exposure has only a small influence on the genetic predisposition to AMD, although individuals with different alleles at the relevant loci might differ from each another. 

There was no gender difference in the amount of retinal damage present in BALB/c retinas compared with B6 retinas after constant light exposure. ¹⁷ However, there was in age-related retinal degeneration. Because the cross we produced was nonreciprocal, all mothers of the F1 mice were BALB/c. This allowed us to see a difference in age-related retinal degeneration between F1 males (all hemizygous for the BALB/c X chromosome and all carrying the B6 Y chromosome) and F1 females. The gender difference carried through to the F2 generation as well. Based on the results of F2 progeny with different X chromosome genotypes, we hypothesized that a gene in a region of the BALB/c X chromosome near the marker DXMit216 conferred some resistance to age-related retinal degeneration, but only in the presence of the B6 Y chromosome. This was deduced from the following: (1) the ONL of retinas from male B6 mice were thicker than those of B6 females; (2) there was no difference in the ONLs between male and female BALB/c mice; (3) F2 males hemizygous for the C allele of DXMit216 were protected from age-related retinal degeneration compared with F2 females homozygous C for the same marker; (4) F2 males hemizygous for the DXMit216 C allele were protected from age-related retinal degeneration compared with F2 males hemizygous for the B6 allele of DXMit216; (5) F2 males hemizygous for the B6 allele for any of the four markers genotyped on the X chromosome appeared to be protected from age-related retinal degeneration compared with F2 females. Further aging studies using F1s from B6 mothers are needed to test this hypothesis. 

In a family study by Klein et al., ¹⁶ with 10 individuals from two generations with AMD inherited in an autosomal dominant fashion, a locus was identified at 1q25-q31. More recently, in a large genome-wide scan study of a cohort of 391 families with a minimum of two individuals with AMD, three categories of disease were established to make the genetics more precise. The result was the identification of four potential AMD loci with LOD scores between 2.0 and 3.16. ¹⁵ The loci were 1q31 (matching the family study), 17q25, 9p13, and 10q26. The ABCA4 gene, which causes several types of autosomal recessive retinal degeneration including Stargardt’s macular dystrophy, has been implicated in AMD. In this case, individuals with damaging mutations in only one allele are thought to be susceptible to the disease. However, the evidence is controversial and, if true, would influence only a small percentage of AMD cases. ^{40 41 42 43 44 45 46 47 48} A second gene implicated in AMD is APOE. The ε4 allele of this gene has been associated with a small protective effect against AMD. ^{49 50 51 52} Ctsd ^tm1Cptr , a disrupted allele of the mouse gene that expresses cathepsin D, produces progressive age-related changes in the mouse retina similar to those of AMD when homozygous. ⁵³ Human chromosomal regions homologous to the three highly significant QTLs are 2p and 3p (Chr 6 QTL), 6q (Chr 10 QTL), and 3q and 21q (Chr 16 QTL). None of the QTLs found in our study are in mouse loci homologous to the AMD-associated human loci or the loci of the AMD-associated genes cited herein. Therefore, these QTLs represent loci of genes that previously, were not known to influence age-related retinal degeneration and may serve as candidates for study in AMD once identified. These same genes may modify the monogenic inherited retinal degenerations as well. 

The mouse age-related retinal degeneration QTLs on Chrs 10 and 16 cover broad regions and come with large 95% confidence intervals (CI) that include many genes. Based on the 2-LOD support intervals shown in Figures 2c and 2e and the MSGCv3 mouse genome sequence map (www.ncbi.nlm.nih.gov/genome/sequence/ provided in the public domain by the National Center for Biotechnology Information, Bethesda, MD), we estimate the distances spanning the QTLs at 30 Mb or more for both the Chr 10 and Chr 16 QTLs. The QTL on mid-Chr 6 has a 2-LOD support interval of approximately 10 cM (Fig. 2a) . Using the bootstrap analysis function from Map Manager QTX (not shown), the 95% CI lies between D6Mit209 and D6Mit284 (∼8.5 cM in our cross), which are placed at 76.4 and 93.4 Mb, respectively on the MSGCv3 map, a distance of 17 Mb. There are well over 200 genes in this region, and many are expressed in retina. Evaluating the many good candidate genes in this Chr 6 QTL region will be assisted by refinement of the locus. To do this, additional studies must be performed with additional F2 progeny from the same intercross, and/or with recombinant inbred C57 x BALB/c (CxB or BxC) strains with “mosaicized” chromosomes, and/or with similar QTLs from other crosses that may overlap. This task is made much easier by the fact that we are starting with a relatively narrow region (for a QTL) because of the strong influence of this Chr 6 gene. Discovery of this and the other QTL genes and their protective alleles may open avenues of study that contribute to the development of gene or pharmaceutical therapies for age-related and other retinal degenerations. 

 Supported in part by NIH Grants EY13280 (MD) and EY01919 and EY02162 (MML), the Foundation Fighting Blindness (MD, MML), Research to Prevent Blindness, That Man May See, Inc., and the Macula Vision Research Foundation (MML). The Center for Inherited Disease Research is fully funded through a federal contract from the National Institutes of Health to The Johns Hopkins University (Contract Number N01-HG-65403). MML is a Research to Prevent Blindness Senior Scientist Investigator.

 Submitted for publication December 6, 2002; revised January 21, 2003; accepted February 13, 2003.

 Disclosure: M. Danciger, None; J. Lyon, None; D. Worrill, None; M.M. LaVail, None; H. Yang, None

 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

 

Figure 1.

 

Thickness of the ONL of the retina of C57BL/6J-c^2J (•) and BALB/cByJ (▪) mice at various times after aging in dim cyclic light. The number of mice at 6, 8, 10, and 12 months were 6, 5, 5, and 3, respectively, for B6 mice, and 6, 5, 3, and 4, respectively, for BALB/c mice.

Table 1.

 

Evaluation and Comparison of Quantitative Trait in Control Mice at 8 Months of Age

Genotype	Average ONL Thickness (μm)	n	Comparison	P *
BALB/cByJ (C)	39.15	30	C vs. B6	8.6 × 10−18
C57BL/6J-c2J (B6)	45.26	23	C vs. F1	4.2 × 10−24
F1	47.30	50	F1 vs. B6	NS (0.12)

Table 2.

 

QTLs from Age-Related Retinal Degeneration Intercross Study

Sig*	Marker(s) at Peak of QTL	cM, † from Centromere	Mb, ‡ from Centromere	LOD Score	% Effect (% Total Genetic Effect), §	Best Fitting Inheritance Model
HS	103 controls	—	—	26.1	69 (100)	Dominant
HS	D6Mit209	33	76.4	13.5	21 (30)	Dominant
	D6Mit284	37	93.4
HS	D10Mit213	11	20.3	6.4	9 (13)	Additive
HS	D16Mit139	43	66.1	7.1	−9 (−13), ∥	Add/recess, ¶
	D16Mit189	55	83.2
Sugg	D14Mit126	5	17.0	2.7	3 (4)	Recessive
Sugg	D18Mit208	38	61.2	2.6	3 (4)	Recessive
Sugg	D12Mit60	16	28.9	2.6	−3 (−4)	Add/dom
	D12Mit236	22	39.5
Sugg	D13Mit19	24	43.4	2.5	3 (4)	Additive
Sugg	D8Mit47	53	106.7	2.1	3 (4)	Add/dom
	D8Mit49	67	124.1

Figure 2.

 

(a, c, e) Interval maps of the LRS (converted to LOD scores) produced by the Map Manager QTX program of the three strong and highly significant quantitative trait loci (QTL). Dashed horizontal lines represent a 2-LOD distance from the peak of the QTL. The vertical projections from the points where the 2-LOD line crosses the graph of the LOD scores conservatively estimate the 95% CI. Each hash mark on the x-axis is 1 cM. (b, d, f) Histograms of the percentage of the total genetic effect compared with the model of inheritance. In addition, the peak markers from this age-related study are shown for the percentage of genetic effect they had in a constant light-induced retinal degeneration study performed previously with the same two strains of mice.¹⁷

Figure 3.

 

Comparison of average ONL thickness scores of F2 progeny of the same genotype for dinucleotide repeat markers on the X chromosome. MC, males hemizygous for the BALB/cByJ allele; MB, males hemizygous for the C57BL/6J-c^2J allele; FC, females homozygous for the BALB/cByJ allele; CB, heterozygous females. (a) Histogram of average ONL thickness scores by genotype. (b) Plots of the negative logarithm of the probability of significant difference determined by the unpaired Student’s t-test between ONL thickness scores in males and females of the same genotype for all four X chromosome markers. (▪) MC versus all females; (□) MB versus all females; (▴) MC versus MB (males); (•) FC versus CB (females).

Figure 4.

 

Average ONL thickness scores in retinas of control mice at each of the 18 measuring positions (3 measurements per position) on the retinal section. (□) C57BL/6J-c^2J, n = 23; (▵) BALB/cByJ, n = 30; (•) F1, n = 50. The numbers (2–5) indicate the posterior superior region where the ONL thickness average was recalculated for the purpose of comparison with a previous light-damage study.

Table 3.

 

Calculation of Age-Related QTLs Based on Average Thickness of ONL 2–5 in the Superior, Posterior Retina

Sig.	QTL	Marker(s) at Peak of QTL	LOD Score	cM from Centromere	Mb from Centromere	% Effect (% Total Genetic Effect)
HS	103 controls	—	25.3	—	—	72 (100)
HS	Chr 6	D6Mit209	9.1	33	76.4	14 (19)
		D6Mit284		37	93.4
HS	Chr 10	D10Mit213	8.0	11	20.3	12 (17)
HS	Chr 16	D16Mit139	7.5	43	66.1	−12 (−17)
		D16Mit189		55	83.2
Sugg	Prox. 14	D14Mit126	2.6	5	17.0	3 (4)
Sugg	Chr 18	D18Mit208	2.1	38	61.2	3 (4)
Sugg	Chr 12	D12Mit60	2.3	16	28.9	−3 (−4)
		D12Mit236		22	39.5
Sugg	Chr 13	D13Mit19	3.0	24	43.4	4 (6)
NS	Chr 8	D8Mit47	—	53	106.7	—
		D8Mit49		67	124.1
Sugg	Chr 9	D9Mit263	2.1	40	76.3	−3 (−4)
Sugg	Dist. 14	D14Mit165	2.0*	52	97.7	3 (4)

The authors thank Ken Manly for his very generous assistance in interpretation of Map Manager QTX analyses and Joseph Jabbra for helping to get the Loyola Marymount vivarium outfitted.