A mouse model for osseous heteroplasia

Abstract

GNAS/Gnas encodes G(s)α that is mainly biallelically expressed but shows imprinted expression in some tissues. In Albright Hereditary Osteodystrophy (AHO) heterozygous loss of function mutations of GNAS can result in ectopic ossification that tends to be superficial and attributable to haploinsufficiency of biallelically expressed G(s)α. Oed-Sml is a point missense mutation in exon 6 of the orthologous mouse locus Gnas. We report here both the late onset ossification and occurrence of benign cutaneous fibroepithelial polyps in Oed-Sml. These phenotypes are seen on both maternal and paternal inheritance of the mutant allele and are therefore due to an effect on biallelically expressed G(s)α. The ossification is confined to subcutaneous tissues and so resembles the ossification observed with AHO. Our mouse model is the first with both subcutaneous ossification and fibroepithelial polyps related to G(s)α deficiency. It is also the first mouse model described with a clinically relevant phenotype associated with a point mutation in G(s)α and may be useful in investigations of the mechanisms of heterotopic bone formation. Together with earlier results, our findings indicate that G(s)α signalling pathways play a vital role in repressing ectopic bone formation.

Figure 1. Protein coding transcript map of the mouse Gnas cluster (modified from ref [66]).

Features of the maternal and paternal alleles are shown above and below the line. Arrows show direction of transcription. Transcripts arising from the first exons of protein coding transcripts, Nesp, Gnasxl, and Gnas splice onto exon 2 of Gnas. For Gnas the unbroken arrow shows predominant maternal expression and the dotted line indicates limited paternal expression. The asterisk indicates the Oed-Sml mutation in exon 6.

Figure 2. Fibromatous skin masses in Sml and Oed mice.

(A) An ulcerated pedunculated hairless mass on the front paw (arrow) of 7-month-old Oed male mouse. (B) X-ray of this lesion shows radio-opacities (arrow). (C) A hairless nodule on the tail (arrow) of a 12-month-old Oed male mouse. (D, E and F) A pedunculated mass in a 8-month-old Oed male mouse. (D) This mass has a narrow pedicel (arrow p) and is focally ulcerated (arrow u). (E) Intact hyperkeratotic epithelium (arrow e) bordering a mass of fibromatous connective tissue (arrow f) with an entrapped follicle (arrow ef) and associated sebaceous gland. (F) Focal areas of osseous heteroplasia (arrow o) within the fibromatous mass (arrow f). Scale bars: A,B,C = 0.5 mm; D = 1 mm; E = 100 µm; F = 50 µm.

Figure 3. Skin radio-opacities increase with age in Sml and Oed mice and correspond to foci of osseous heteroplasia.

(A) X-ray image of radio-opacities in the skin of a 12-month-old Sml male imaged on a 1×1 cm grid. (B) The number and (C) cumulative area of skin radio-opacities in Sml and Oed male mice at 6 & 9 months of age are not significantly different from +/+ controls, however they are significantly increased in the 12 & 15-month-old cohorts compared to +/+ mice. (D) The number and (E) cumulative area of skin radio-opacities in Sml and Oed females at 6 & 9 months of age are not significantly different from +/+ controls, however they are significantly increased in the 12 & 15-month-old Sml compared to +/+ controls. In the box and whisker plots each box represents the median with 25 and 75% inter-quartile ranges, with whiskers representing the data range (minimum and maximum). The group size n is indicated alongside each histogram bar, whereas the circled numbers are the off-scale maximum values. ns = not significant P>0.05, * P<0.05, **P<0.01, ***P<0.001. Kruskall Wallis non-parametric one-way ANOVA tests were performed with Dunn's multiple comparison tests for post hoc testing. † A single male +/+ control with high levels of radio-opacities was an outlier that had a wounded skin. (F). Radio-opacities correspond to foci of osseous heteroplasia (arrow o) with osteoclasts (arrow oc) beneath intact skin. (G) Focus of hematopoietic tissue (arrow h) within an area of osseous heteroplasia, inset is enlarged view (arrow h), the bone surface has an osteoblast layer (arrow ob). Scale bars: F = 100 µm; G = 200 µm.

Figure 4. Plasma biochemistry phenotypes of male Sml and Oed mice.

(A) Plasma PTH was significantly elevated in Oed mice at 6 & 9 and 12 & 15 months of age compared to +/+ controls. The modest elevation in Sml PTH levels at 6 & 9 months failed to reach significance (P = 0.053) but PTH was significantly elevated at 12 & 15 months compared with respective +/+ controls. Plasma calcium corrected for albumin levels (B) in Oed mice was lower than their respective +/+ controls at 6 & 9 months but not at 12 & 15 months. Calcium in Sml males was not lower than in their respective +/+ controls at 6 & 9 months but was lower than controls at 12 & 15 months. Plasma phosphate levels (C) in Oed mice were higher compared with their respective +/+ controls at 6 & 9 months but not at 12 & 15 months. Plasma phosphate in Sml males was not significantly different from +/+ controls at 6 & 9 months and at 12 & 15 months was lower than +/+ controls. The group size n is indicated alongside each histogram bar, the error bar is ± s.e.m; ns = not significant P>0.05, *P<0.05, **P<0.01, ***P<0.001. 2-tailed t-tests with unequal group variance were performed on the PTH data.