Sharkey, a new mutation in the Sostdc1 gene

Jill Giggey, Michelle M. Curtain, Leah Rae Donahue

Source of Support: NIH-NEI grant EY015073 to Leah Rae Donahue

Mutation (allele) symbol: shk

Mutation (allele) name: sharkey

Gene symbol: Sostdc1

Current strain name: B6(NOD) H2^g7-Sostdc1^shk/J

Stock#: 005717

Phenotype categories: teeth, hearing

Origin and description

This recessive mutation arose spontaneously in 2003 in one of The Jackson Laboratory research colonies. It arose on a predominantly C57BL/6J strain that was congenic for the NOD-derived H2^g7 and for a T Cell Receptor transgene with an unmapped insertion site. The transgene was removed from this strain, but H2^g7 remains.

Mice homozygous for the sharkey mutation display supernumerary incisors. (See Photo) The colony is maintained by sibling matings where either the male or female is a homozygote and the other is a heterozygote.

Pathology

Using our standard pathological screen tissues from three male mutants and one male heterozygote were prepared for histopathological examination. The screen revealed no lesions in any somatic tissues of two mutants ages 4 and 17 weeks old. A 15-week-old mutant had otitis media. A closer exam of the teeth in the mutants revealed that there are separate roots for these extra teeth. One mutant and one control were assessed for hearing loss by auditory brainstem response (ABR). The four-month-old mutant had severe hearing loss whereas the control had normal hearing. A one-year-old mutant was deaf and a littermate control had normal ABR thresholds. A clinical eye exam revealed no abnormalities in either mutants or controls.

Genetic analysis

A cross was performed between a homozygous sharkey female and a CAST/Ei male. F1 progeny were intercrossed and 59 mutant F2 animals were genotyped for mapping studies. Using our standard mapping protocol the mutation was mapped to a region on Chromosome 12 between D12Mit146 and D12Mit271where the Sostdc1gene is located.

PCR primers were designed to amplify each exon of the Sostdc1 gene using IDT Technologies. PCR products were sequenced at The Jackson Laboratory core sequencing facility. The sequence of each exon was then compared to the mouse sequence publicly available at ExonPrimer.html. A base pair deletion was detected in exon 2 of the Sostdc1 gene resulting in a frame shift mutation with a premature stop codon. (See Chromatogram)

Biological Characterization

Looking at eight homozygote by heteozygote matings, 20 mice out of 39 were mutants, just over the 50% expected ratio. Strict homozygote matings produce all affected pups therefore penetrance is 100%. All mutants have supernumerary incisors, specifically an extra pair of upper and lower incisors that erupt posterior to the normal incisors. But there is variability in the phenotype. Some mutants’ supernumerary incisors grow slower while others exhibit faster growth and therefore need to have their teeth trimmed back more frequently. When this strain was crossed to CAST/Ei for mapping, affected mice from the cross only got upper supernumerary incisors indicative of modifiers specific to strain background.

The upper palate also displayed unusual patterning. The control had symmetrical patterning while the mutants had different degrees of disorganized formation.

A. DEXA Analysis of Whole Body BMD and Body Composition

DEXA analyses were performed using the PIXImus densitometer (PIXImus, LUNAR, Madison, WI) Skeletal provides skeletal and body composition values including Bone Mineral Density (BMD, g/cm²), Bone Mineral Content (BMC, g/cm²), total body mass (g), lean mass (g), fat mass (g), and % fat were collected for six male and six female mutants and controls were collected at twelve weeks of age. (See Table 1) Values for homozygous sharkey mice were not different for either males or females. However, there were significant differences in sharkey control males compared to female controls with whole body BMD, BMC, lean, total mass and Skull BMD/Body BMD. And while there are differences between sharkey male and female mutants, none were significant. (See Graph 2 and Graph 3 ). Overall, the differences seem based on sex in the controls and that sharkey mutants grow normally to twelve weeks of age in spite of repeated tooth trimming in some of the mutants. Normal grain was supplemented with ground diet of the same formulation.

B. Skeletal and Craniofacial Morphology

Skulls of six male and six female homozygous mutants and six male and female 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 were also made using digital calipers (Stoelting, Wood Dale, Ill) with previously established landmarks. (Table 2) Female sharkey mutants had significantly shorter skull height, lower jaw length, and less height to length and height to width ratios than controls. Only skull width differed in sharkey males compared to controls. Gender differences were significant with the sharkey mutation only for the ratio between upper and lower jaw where the ratio was greater in females. In controls, males had longer and wider skulls and longer lower jaws. (See Graph 1 and Graph 2 )

Acknowledgements

We would like to thank Bob Giggey for originally identifying the mutant, Heping Yu for ABR analysis, Norm Hawes for clinical eye evaluation, Coleen Marden and Rod Bronson for pathological screening and Leona Gagnon for her guidance in molecular analyses.

The Jackson Laboratory