A New Spontaneous Mutation Causing Corneal Opacity

Jill Giggey, Joiel Bauschatz, Michelle Curtain, Julie Hurd, Norman Hawes, Bo Chang and Leah Rae Donahue, Ph.D.

Source of Support: This research was supported by two grants awarded to The Jackson Laboratory by The National Eye Institute, one to Dr. Leah Rae Donahue entitled "Gene Discovery For Craniofacial Disorders" (RO1 EY015073) and one to Dr. Bo Chang entitled "Mouse Models of Human Hereditary Eye Diseases" (RO1 EY07758).

Mutation (allele) symbol: whe

Mutation (allele) name: white eyes

Gene symbol: whe

Strain of origin: B6.V-Lep^ob/J

Current strain name: B6.V-Lep^ob-whe/J

Stock #: 005624

Origin and Description

We report a new spontaneous, recessive mutation, named "white eyes" (whe), which arose at The Jackson Laboratory in 2003, in the B6.V-Lep^ob/J colony. The homozygous whe mutation usually is expressed bilaterally and is characterized by a small, white spot on the cornea; occasionally, only one eye will be affected. When viewed with a slit lamp and by indirect ophthalmoscope, the eye displays white spots and an atypical growth of blood vessels on the cornea.

Since the obese phenotype, characteristic of Lep^ob/ Lep^ob homozygotes of the B6.V-Lep^ob/J strain, continues to segregate in the whe colony, the stock is maintained by mating a non-obese male or female whe/whe to a non-obese mouse that is heterozygous for whe. Both non-obese males and non-obese females are fertile; females have normal litter sizes and lactate normally. The strain was tested for penetrance by mating two whe homozygotes and was found to be 100% penetrant (51 pups out of 51 born were affected). The whe/whe mutation was mapped to Chromosome 11 with 2.5% recombination at marker D11Mit71 which is located 6.8 Mb (Ensembl Genome Browser, v33) from the centromere.

Genetic Analysis

White eyes is inherited as a recessive mutation as shown by traditional breeding experiments performed as part of the linkage cross for genetic mapping. For linkage analysis, a C3H/HeJ (JR001912) male, without retinal degeneration, was mated to a homozygous whe/whe female. F1 hybrids were then intercrossed to produce F2 progeny. There were no visible mutants seen in the F1 generation (0/39) and 21.9% of F2 progeny were mutants (32/146). F2 progeny were observed for the white, opaque color of the eye, and an additional screening of the cornea with a slit lamp and indirect ophthalmoscope was performed to confirm the mutant phenotype of affected mice. Spleen and tail tip of mutant mice were collected and stored at -70° C for subsequent DNA typing to map the mutation. DNA was extracted from the tail tips by a standard hot sodium and Tris (HotSHOT) procedure (Truett, et al., 2000), and polymerase chain reaction was carried out with MIT primer pairs (MapPairs, Research Genetics, Huntsville Ala.).

Because of the whe eye phenotype, we began by testing for linkage (using pooled DNA from affected F2s) with Pax6 on Chromosome (Chr) 2, Tgfa on Chr 6, Egfr^wa on Chr 11, and Bmp4 on Chr 14. No linkage was observed at these four locations. Linkage of white eyes was first detected on Chr 11 at 8.8 Mb from the centromere with marker D11Mit226 (Ensembl Genome Browser, v33). Twenty-one DNA samples were then typed individually and results showed linkage with 2.5% recombination at marker D11Mit71, 6.8 Mb from the centromere (Ensembl Genome Browser, v33). A spontaneous mutation called "gaping lids" is located on Chr 11 at 0.2 cM from the centromere, and homozygotes for this mutation display a phenotype similar to whe; however, the gapping lids mutant was not available for a complementation test. Although not confirmed to be the causative gene for gapping lids, Neurofibromin 2 (Nf2) found at 0.25 cM was considered a candidate gene (Juriloff, personal communication). Due to the similarity of phenotypes and its chromosomal location, we sequenced the coding regions of Nf2, but found no mutation of Nf2 in DNA from whe homozygotes.

Biological Characterization

A. DEXA Analysis of Whole Body aBMD and Body Composition

Whole body areal bone mineral density (aBMD), bone mineral content (BMC) and body composition (lean, fat, and %fat mass) was assessed by PIXImus densitometry (GE LUNAR, Madison, WI, see protocol on this website). No significant differences were found between mutants and controls in either gender (Table 1).

Table 1: PIXImus Densitometric Measurements of Twelve Week Old Mutant and Control Mice of Both Sexes (n=5, mean±SEM).

B. Craniofacial Morphology

Skulls of six male and six female mutants and controls were collected at twelve weeks of age, prepared by incomplete maceration in potassium hydroxide, stained with alizarin red, and stored in undiluted glycerin (Green, 1952). Morphological measurements of the skull (Table 2) were made using digital calipers (Stoelting, Wood Dale, Ill) with previously established landmarks (Richtsmeier, 2000, see protocol this website). Inner canthal distance (Figure 1) and lower jaw length (Figure 2) were significantly different in whe/whe females compared to control females. No other skull measurements or skull measurement ratios were significantly different for either gender when homozygotes were compared to controls.

Table 2: Digital Caliper Measurements of Twelve Week Old

Skulls of Male and Female whe/whe and Control Mice

(n=6, mean±SEM; a=p value <0.05).

Figure 1: Mean Inner Canthal Distance of Male and Female

whe/whe and Control Mice (n=6).

Figure 2: Mean Lower Jaw Length of Male and Female

whe/whe and Control Mice (n=6).

C. Hearing Tests

Hearing was assessed by ABR threshold analysis (Zheng et al. 1999) using two mutants and two controls of each gender at three months of age. The ABR results showed that both mutant and control mice have normal hearing.

D. Eye Examination

Eight mutants at four weeks of age were examined by a slit lamp and indirect ophthalmoscope. Results showed that the mice do not have a cataract. In all the mutants, the cornea has a white vascular area. Eye histology shows a vascular cornea and attachment of the cornea to the iris (synechia). The retina and lens appear normal in heterozygotes and in most homozygotes. One out of eight homozygotes showed an outer nuclear layer with rosettes and degeneration.

Figure 3: The eye appearance of whe mutant mice.

(A) The normal control shows a closed eye at 2 days of age.

(B) An open eye is shown in a whe/whe mouse at 2 days of age.

(C) A cornea opacity and a white vascular area are shown

in a whe/whe mouse at 5 months of age.

E. Pathology

One male mutant was perfused at nineteen weeks of age via cardiac infusion of Bouin's fixative following admission of anesthesia. Testicular atrophy was observed and osteoarthritis was noted in both hips of a female mutant perfused at fifty-four weeks of age.

Discussion

In normal human development, the eyes close during the eighth week of gestation and reopen at the seventh month of gestation (Juriloff, 2000). During normal development in the mouse, the eyelids grow, flatten across the eye and fuse with each other between days 14 and 16 of gestation and open approximately 14 days after birth (Harris, 1982 and Juriloff, 2000). When the eyelids do not fuse properly in the mouse, the eyes are open at birth and cause corneal opacity or degenerative changes of the eye when mice reach adulthood (Juriloff, 2000).

In the whe mutation reported here, the eyelid is either completely or partially open when neonates are two days old. Consequently, an infection of the eye develops and a characteristic white spot is present on one or both eyes. Further investigation of the mutant eye shows an atypical growth of blood vessels on the cornea. Histology shows cornea inflammation and iris and corneal attachment. This new mutation is a valuable model of abnormal eye development, even though the causative gene has not been identified.

Acknowledgements

We thank the following for their respective contributions to this work: Lisa DeLaittre, discoverer of the original mutant; Coleen Marden, preparation of tissues for histological assessment; Rod Bronson, Ph.D, pathological evaluation; Pat Ward-Bailey, genetic analysis; Jane Maynard, tissue preparation for mapping study; Heping Yu and Qing Yin Zheng, evaluation of ears.

References

Juriloff DM, Harris MJ, Banks KG and Mah DG (2000). Gaping Lids, gp, a mutation on centromeric Chromosome 11 that causes defective eyelid development in mice. Mouse Genome 11(6): 440-7.

Harris, MJ, and McLeod MJ (1982). Eyelid Growth and Fusion in Fetal Mice. A Scanning Electron Microscope Study. Anatomy and Embryology 164: 207-220.

Mouse Genome Database (MGD) Mouse Genome Informatics Project, The Jackson Laboratory, Bar Harbor, Maine. World Wide Web (URL: http://www.informatics.jax.org).

Truett GE, Heeger P, Mynatt RL, Truett AA, Walker JA, and Warman ML (2000). Preparation of PCR-Quality Mouse Genomic DNA with Hot Sodium Hydroxide and Tris (HotSHOT). Biotechniques 29:52-54.

Zheng QY, Johnson KR, Erway LC (1999). Assessment of hearing in 80 inbred strains of mice by ABR threshold analyses. Hear Res 130, 94-107.

The Jackson Laboratory