Mapping the Bst mutation on mouse Chromosome 16: a model for human optic atrophy

D. S. Rice*, R. W. Williams*, P. Ward-Bailey#, K. R. Johnson#, B. S. Harris#, M. T. Davisson#, and D. Goldowitz*

*The University of Tennessee, Center for Neuroscience and Department of Anatomy and Neurobiology, 875 Monroe Avenue, Memphis, TN 38163, #The Jackson Laboratory, Bar Harbor, Maine 04609, USA


Autosomal dominant optic atrophy (OPA1) is the most common form of hereditary optic atrophies in humans, with a frequency of 1:50,000 (165500; GDB). A recent study has localized OPA1 to Chromosome (Chr) 3 between q28-qter (Eiberg et al. 1994). OPA1 is characterized by a loss of visual acuity, deficits in color vision, and scotomas of varying size (Eliott et al. 1993). Retinas of patients with OPA1 have a reduction in the number of retinal ganglion cells and a decrease in myelin content in the optic nerves, chiasm, and tracts (Johnston et al. 1979; Kjer et al. 1983). Neurons that are in the main target of the retinal ganglion cell projection, the dorsal lateral geniculate nucleus, are also atrophic (Kjer et al. 1983). Other cell populations in the retina of humans with OPA1 appear to be normal (Johnston et al. 1979; Kjer et al. 1983). OPA1 is a dominant mutation, but the expression of the phenotype is highly variable both within and among families (Kline and Glaser 1979). The loss of visual acuity and the atrophy of the optic nerves often varies between right and left sides (Kline and Glaser 1979; Kjer et al. 1983).

Recently, we have identified a striking abnormality in optic nerves of mice that are heterozygous for the spontaneous mutation belly spot and tail (Bst). Bst is a semi-dominant, homozygous lethal mutation that arose in the inbred strain C57BLKS (BKS; previously denoted C57BL/Ks). Heterozygous mice have a kinky tail, white feet, and a white spot at the ventral midline. In approximately 50% of the Bst/+ mice, there is a reduction or a complete absence of the pupillary light reflex in one or both eyes (Rice et al. 1993). This neurological phenotype is associated with a unilateral or bilateral atrophy of the optic nerves. As in humans with OPA1, the severity of the atrophy of the optic nerves is highly variable—ranging from a slight reduction in the number of ganglion cell axons in one optic nerve to a complete elimination of both optic nerves. The surface area of the retina and the appearance of the inner and outer nuclear layers are qualitatively normal (Rice et al. 1993).

The Bst locus has been mapped previously as the distal-most locus of a three-point cross in relation to Igl1 (immunoglobin lambda-1) and md (mohaganoid) on Chr 16 (Epstein et al. 1986). Harris et al. (1989) subsequently mapped Bst in a two-point cross with Sod1 (superoxide dismutase-1). Collectively, these studies place the Bst locus 25 to 42 cM distal to the centromere and proximal to Sod1. This region of mouse Chr 16 is conserved in human Chr 3 (Reeves and Citron 1994). Given the marked phenotypic similarity of retinal phenotypes between OPA1 and Bst and the chromosomal homology, we have generated a higher resolution map of Bst on Chr 16 using an intraspecific backcross. F1 hybrids were produced by crossing BKS-Bst/+ females to AKR males. The (BKS x AKR)F1-Bst/+ males and females were crossed to wildtype BKS, generating a total of 157 backcross progeny. These progeny were phenotyped for Bst by inspecting the tail for kinks and the belly for white hairs. Genomic DNA was isolated for the analysis of simple sequence length polymorphisms (SSLP). A total of 11 primer pairs recognizing SSLP loci were used to map Bst more precisely. PCR reactions were carried out as described by Dietrich et al. (1992) with two modifications: 1) the Taq DNA polymerase concentration was doubled (.5U/rxn), and 2) 30, instead of 25 cycles, were run. PCR products were separated on 7% non-denaturing polyacrylamide gels and stained with ethidium bromide. Recombination frequencies were analyzed using the program Map Manager v2.51(Manly and Elliott 1991).

The results of the haplotype analysis establish the following order among loci with distances in cM ± standard error: D16Mit87-21.1 ± 6.6-D16Mit110-1.3 ± 1.3-D16Mit11-5.1 ± 1.8-D16Mit138, D16Mit84, D16Mit39-1.3 ± 0.9-D16Mit168-1.9 ± 1.1-Bst-6.4 ± 2.0-D16Mit140, D16Mit174-1.3 ± 0.9-D16Mit114-3.2 ± 1.4-D16Mit94. Thus, according to the placement of these markers on the consensus map for mouse Chr 16 (Reeves and Citron, 1994), Bst lies approximately 39 cM from the centromere (Fig. 1).

The percentage of mutants in the backcross (33.1%) is less than the 50% expected for a dominant Mendelian trait. There are two possible explanations. First, the penetrance of the mutation may be decreased in the backcross progeny (see Epstein et al. 1986). If the paucity of Bst/+ mutants in the backcross is reduced due to incomplete penetrance, then flanking markers should still exhibit normal 1:1 segregation. However in our backcross, markers linked to the Bst locus exhibit a marked segregation distortion, consistent with our Bst genotype assignments (Fig. 2). Second, the in utero survival rate of the Bst heterozygote may be compromised. The mean litter size is reduced in the backcross compared to the intercross (6.0 vs. 7.8), and the percentage of Bst/+ mutants is reduced in backcross litters (Table 1). Furthermore, Bst heterozygotes on the C57BLKS inbred background are not as viable (mean litter size = 4.6) as heterozygous mice whose genetic background is a mosaic of AKR and C57BLKS alleles. Thus, the disparity in the BKS alleles in the backcross is probably the result of the in utero elimination of Bst/+ mutants. In a related study, we have found in utero death associated with a high incidence (about 30%) of exencephaly in crosses using Bst/+ mice (Rice et al. 1993).

Haplotype data for Bst locus

We have mapped the Bst locus to a region of mouse Chr 16 that is conserved in human Chr 3. Some of the loci on human Chr 3 that are also located on mouse Chr 16 include: apolipoprotein d (APOD), pituitary transcription factor, (PIT1), dopamine D3 receptor (DRD3), preprosomatostatin (SMST), stefin 1 (STF1), ROS2, and growth-associated protein (GAP43; Fig. 1). Gap43 has recently been shown to be important for the growth of retinal ganglion cell axons at the optic chiasm in the mouse (Strittmatter et al. 1995). However, based on the current map positions, GAP43 (which maps to human Chr 3q13.1-q13.2; Naylor et al. 1994) does not appear to be a candidate for OPA1 although it cannot be ruled out as a candidate for the Bst mutation.

OPA1 and Bst have many similarities. Both mutations are inherited as dominant phenotypes with variable expressivity, and both appear to target retinal ganglion cells. It has been postulated that the OPA1 mutation results in a primary degeneration of the cells in the ganglion cell layer and, subsequently, in atrophy of the optic nerves (Kjer et al. 1983). The decrease in retinal ganglion cells in the Bst/+ mouse is evident as early as the day of birth (Rice et al. 1993) and before the onset of naturally occurring ganglion cell death (Williams et al. 1990).

The mechanisms responsible for the congenital decrease in ganglion cell number in humans with the OPA1 mutation and in Bst/+ mutant mice are unknown. Comparison of the order of homologous loci in the mouse and human chromosomal maps for this region suggests that OPA1 and Bst map to different regions of the conserved segment. Therefore, they either may not be mutations in the same gene or the gene order may differ between the mouse and human chromosome within these conserved segments. Despite this caveat, the remarkable similarities in the atrophy of the ganglion cell population between OPA1 and Bst make the Bst mutant mouse a good model to study the abnormal development of the ganglion cell population.

Acknowledgments. We would like to thank Janice Hyatt for assistance with genomic DNA isolation. This work was supported by National Institutes of Health (NIH) Grant NS EY-09586 (DG), NIH Neuroscience training grant NS-07323 (DSR), NIH Grant PO1 RR01183, and Cancer Core Grant CA34196 (BSH, PWB, KRJ, MTD).


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Figure Legends

Fig. 1. Genetic map of mouse Chr 16. The distances given to the left of the map are based on the recombination estimates of the D16Mit loci typed in the (BKS x AKR) F1-Bst/+ x BKS intraspecific backcross. Loci that are underlined have been mapped on mouse Chr 16 and human Chr 3. Map positions of these loci were taken from the Chr 16 committee report (Reeves and Citron 1994; MGD), the Portable Dictionary of the Mouse Genome (Williams, 1994), or the Human Genome Database (GDB). Their positions on human Chr 3 are given to the left of the map.

Fig. 2. Haplotype data for the intraspecific backcross showing the markers flanking the Bst locus segregating on Chr 16. There is a marked segregation distortion ratio in the flanking markers. The number of backcross progeny with each haplotype is identified by the number beneath the columns. Genotypes are designated by the black (BKS) and white (AKR) squares.

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