A New Semi-Dominant Short Nose Mutation

Jill M. Giggey, Michelle M. Curtain, Julie M. Hurd,

Joiel D. Bauschatz and Leah Rae Donahue, Ph.D. 

Source of Support: This research was supported by a grant awarded to The Jackson Laboratory by the National Eye Institute titled "Gene Discovery For Craniofacial Disorders" (RO1 EY 015073 - Dr. Leah Rae Donahue, PI).

Strain of Origin: C57BL/6J-Nm2885

Phenotype Category: Craniofacial

Discoverer: Louise Dionne

Origin and Description

We report here a new semi-dominant mutation that was discovered in the C57BL/6J strain in the (ENU) Neuroscience Mutagenesis Facility (NMF) at The Jackson Laboratory in 2000. The semi-dominant mutation is characterized by a small, short nose and round skull with wide set eyes. The stock is currently maintained by mating a heterozygous male to an unaffected female littermate. Both males and females are fertile; heterozygous females have small litters, approximately 2-5 pups per litter. Weekly trimming of teeth is required due to misalignment of teeth causing malocclusion. The chromosomal location of this short nose mutation has not been determined, but two loci have been ruled out as candidates. DNA was tested for markers on Chromosome 4 (D4Mit203, D4Mit338, D4Mit336), because of the similarity in phenotype seen between this new mutation and snub-nose like (sno) and achondroplasia (cn) (Mouse Genome Database, TJL). Results showed high recombination frequencies indicating no linkage at these locations.

Biological Characterization

A. DEXA Analysis of Whole Body Bone Mineral Density (BMD)

Whole body weight, whole body BMD and whole body Bone Mineral Content (BMC) assessed by PIXImus densitometry (GE LUNAR, Madison, WI) were not significantly different between mutants and controls in either gender (Table 1). Whole body fat and percent fat between female mutants and controls were statistically significant (Figures 1 and 2), but were not significant between male mutants and controls. Whole body lean was not statistically significant between mutant and control of either gender. Lastly, skull BMD and skull BMC were not significantly different between male and female mutants and controls.

Table 1: PIXImus Densitometric Measurements of Twelve Week Old C57BL/6J-Nm2885

Nm/+ and Control Males and Females (n=5, mean+/-SEM).

*p-value <0.05 between mutant and control

Figure 1: Whole Body Fat in C57BL/6J-Nm2885 Male and Female Mutant Mice

Compared to Controls (n=6).

*Statistically significant

Figure 2: Percent Fat in C57BL/6J-Nm2885 Male and Female Mutant Mice

Compared to Controls (n=6).

*Statistically significant

B. Craniofacial Morphology

Skulls of five male and five female mutants and sex-matched 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). During the collection process, right ear pinnae were measured (Table 2) with digital hand calipers (Stoelting, Wood Dale, I11). Morphological measurements of the skull (Table 2) were also made using digital calipers (Stoelting, Wood Dale, Ill) with previously established landmarks (Richtsmeier, 2000). Skull length, nose length and upper jaw length were significantly different between male mutants and controls, but were not different in females (Figures 3-5). Inner canthal distance was statistically different in both genders between mutants and controls (Figure 6). Skull height and skull width measurements were not significantly different in male or female mutants and controls. Skull measurements for male and female mutants were compared and there were no statistical differences found.

Ratios for the skull measurements were calculated. In males, the upper jaw to lower jaw length ratio, skull height to skull length ratio, and the skull length to skull width ratio were all statistically different (Figures 7-9). However, the skull length to nose length ratio and the skull height to skull width ratio were not statistically significant in males. All of the ratios for skull measurements in females were not different when mutants were compared to controls. Lastly, there were no differences found between male and female mutants and controls with right ear pinnae measurements. 

Table 2 : Digital Caliper Measurements of Twelve Week C57BL/6J-Nm2885 Nm/+ and Control Male and Female skulls stained with Alizarin Red (n=5, mean+/-SEM).

*p-value <0.05 between mutant and control. 

Figure 3: Skull Length in C57BL/6J-N2885 Male and Female Mutant Mice

Compared to Controls (n=6).

*Statistically significant

Figure 4: Nose Length in C57BL/6J-Nm2885 Male and Female Mutant Mice

Compared to Controls (n=6).

*Statistically significant

Figure 5: Upper Jaw Length in C57BL/6J-Nm2885 Male and Female Mutant Mice

Compared to Controls (n=6).

*Statistically significant

Figure 6: Inner Canthal Distance in C57BL/6J-Nm2885 Male and Female Mutant Mice

Compared to Controls (n=6).

*Statistically significant

Figure 7: Jaw Length Ratio in C57BL/6J-Nm2885 Male and Female Mutant Mice

Compared to Controls (n=6).

*Statistically significant

Figure 8: Skull Height to Skull Length Ratio in C57BL/6J-Nm2885 Male and Female Mutant Mice

Compared to Controls (n=6).

*Statistically significant

Figure 9: Skull Length to Skull Width Ratio in C57BL/6J-Nm2885 Male and Female Mutant Mice

Compared to Controls (n=6).

*Statistically significant

C. Hearing Tests

Hearing was assessed by ABR threshold analysis (Zheng et al, 1999) with three mutants at ages six, seven and eight months, respectively. All mutants had severely impaired hearing at low frequencies and were completely deaf at low frequencies. All controls had normal hearing.

D. Eye Examination

One mutant and one control from each gender were examined by a slit lamp and indirect ophthalmoscope. The male mutant had which is common in the C57BL/6J background strain and the female mutant had a large retinal coloboma, which is a developmental defect seen at birth. Both controls had normal eyes. 

Pathology

One male mutant was perfused at 11 weeks of age via cardiac infusion of Bouin's fixative following admission of anesthesia. The ear did not develop correctly and there was otitis media. Results indicated the incus or malleus was still embedded in the connective tissue. We also found dilation in the lymphatics, more specifically in the thymus.

A mutant at six months old was perfused and otitis media was present in both ears and macrophages were found within the eustachian tube. The inner hair cells looked normal but the ganglion cells were degenerate.

Discussion

We have a new short nose, round skull and wide set eyes mutation that arose on the C57BL/6J strain in the ENU program here at The Jackson Laboratory. DEXA data showed a statistical difference in whole body fat and percent fat between female mutants and controls but not between male mutants and controls. Female mutants had higher whole body fat and percent fat than controls. All other data was not significant among mutants and controls of either gender. The raw data indicates male mutants have less lean and whole body fat than controls. However, female controls have less lean mass than mutants.

Digital caliper measurements of the skull showed a statistical difference in five out of the seven established skull measurements, including skull length, nose length, inner canthal distance and upper jaw length among male mutants and controls. In females, there was one skull measurement found to be statistically different, the inner canthal distance. Therefore, the males display a short nose and round skull, as illustrated by a shorter skull length, nose length and upper jaw length, as well as, wide set eyes, demonstrated by an extended inner canthal distance. Statistically, female mutants exhibit wide set eyes. The raw data shows female mutants have a shorter skull length, nose length, a wider skull width and a shorter upper jaw but a longer lower jaw. Maloclusion is common in this strain due to the differences found with the upper and lower jaw lengths. In conclusion, male mutants have a more severe craniofacial dysmorphology than female mutants compared to controls and there is high variability within the strain between male and female mutants and controls.

In humans, a craniofacial disorder called Apert syndrome has similar characteristics to our mice, such as mid-face hypoplasia, shorter upper jaw, dental abnormalities and hearing loss. Children with this syndrome exhibit a sunken middle face, bulging wide set eyes, a beaked nose and an underdeveloped upper jaw leading to crowded teeth and other dental problems (John Hopkins University website). The discovery of this gene will be useful in studies of mid-face hypoplasia, hearing loss and dentition and will provide a useful mouse model for the scientific community.

Acknowledgements

We thank the following for their excellent expertise:

Louise Dionne, Discoverer
Colleen Marden, Pathology
Rod Bronson, Ph.D, Pathology Evaluation
Pat Ward-Bailey, Genetic Analysis
Jane Maynard, Tissue Preparation
Norm Hawes, Evaluation of Eyes
Heping Yu and Qing Yin Zheng, Evaluation of Ears 

References

Green, M.C. (1952) A rapid method for clearing and staining specimens for the demostration of bone. The Ohio Journal of Science 52(1):31-33. January 1952.
Mouse Genome Database (MGD) Mouse Genome Informatics Project, The Jackson Laboratory, Bar Harbor, Maine.
Richtsmeier JT, Baxter LL, and Reeves RH. (2000) Parallels of craniofacial maldevelopment in Down Syndrome and Ts65Dn mice. Dev. Dyn. Feb;217(2): 137-45.
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.