Histological analysis of skin x-ray radio-opacities in <i>Oed</i> and <i>Sml</i> mice.

<p>2×2 Fisher Exact tests were used to compare proportions of <i>Sml</i> or <i>Oed</i> with +/+ control mice bearing each lesion. ns = not significant <i>P</i>>0.05,</p>*<p><i>P</i><0.05,</p>***<p><i>P</i><0.001.</p

Fibromatous skin masses in <i>Sml</i> and <i>Oed</i> mice.

<p>(A) An ulcerated pedunculated hairless mass on the front paw (arrow) of 7-month-old <i>Oed</i> 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 <i>Oed</i> male mouse. (D, E and F) A pedunculated mass in a 8-month-old <i>Oed</i> 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.</p

Skin radio-opacities increase with age in <i>Sml</i> and <i>Oed</i> mice and correspond to foci of osseous heteroplasia.

<p>(A) X-ray image of radio-opacities in the skin of a 12-month-old <i>Sml</i> male imaged on a 1×1 cm grid. (B) The number and (C) cumulative area of skin radio-opacities in <i>Sml</i> and <i>Oed</i> 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 <i>Sml</i> and <i>Oed</i> females at 6 & 9 months of age are not significantly different from +/+ controls, however they are significantly increased in the 12 & 15-month-old <i>Sml</i> 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 <i>n</i> is indicated alongside each histogram bar, whereas the circled numbers are the off-scale maximum values. ns = not significant <i>P</i>>0.05, * <i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><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.</p

Plasma biochemistry phenotypes of male <i>Sml</i> and <i>Oed</i> mice.

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

Protein coding transcript map of the mouse <i>Gnas</i> cluster (modified from ref [<b>66</b>]).

<p>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, <i>Nesp</i>, <i>Gnasxl</i>, and <i>Gnas</i> splice onto exon 2 of <i>Gnas</i>. For <i>Gnas</i> the unbroken arrow shows predominant maternal expression and the dotted line indicates limited paternal expression. The asterisk indicates the <i>Oed-Sml</i> mutation in exon 6.</p

<i>GALNT3</i> mutations identified in familial tumoural calcinosis (FTC) and hyperostosis-hyperphosphataemia syndrome (HHS) patients.

a<p>The locations of these mutations are illustrated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043205#pone-0043205-g001" target="_blank">Figure 1</a>.</p>b<p>Nucleic acid change at base pair in the cDNA sequence (Genbank accession number NM_004482.3); del, deletion; skip, exon skipping; and ins, insertion.</p>c<p>aa, amino acid; del, deletion; fs, frameshift. Mutations 2, 4, 5, 6, 14, 15, 16, 17, 18, 20, 21, 24 and 25 were identified as homozygotes. Of these, mutations 5, 14, 17, 21 and 25 were also identified in other patients as compound heterozygotes. Compound heterozygous mutations were identified in the following combinations: 1+9, 3+5, 3+21, 7+23, 8+12, 10+14, 11+19, and 17+25.</p>d<p>FTC, familial tumoural calcinosis; HHS, hyperostosis-hyperphosphataemia syndrome.</p

Mislocalization and defective glycosylation of mutant Galnt3.

<p>(<b>A</b>) COS-7 cells were transiently transfected with either EGFP-wild-type, WT (Trp589), or EGFP-mutant (Arg589) constructs, and counterstained with anti-GM130 antibody, which immunostains the Golgi apparatus (red), or anti-PDI antibody, which immunostains the ER (red). DAPI was used to stain the nucleus (blue). WT Galnt3 co-localizes with GM130, but not PDI (data not shown), thereby revealing that it is targeted to the Golgi apparatus. However, the mutant Galnt3 co-localizes with PDI and is predominantly found in the ER. (<b>B</b>) Western blot analysis of kidney homogenates using anti-GALNT3 antibody, revealed that protein lysates from WT littermates, and <i>Tcal</i>/+ mice had three immunoreactive products (a, b and c) whereas those from <i>Tcal/Tcal</i> mice had only two products (b and c). PNGase F treatment resulted in loss of the largest Galnt3 product (band a) observed in the lysates from WT littermates, and <i>Tcal</i>/+ mice, indicating that these were glycosylated products.</p

Mapping of <i>Tcal</i> locus and identification of <i>Galnt3</i> mutation.

<p>(<b>A</b>)The <i>Tcal</i> locus, which originated in a C57BL/6 ENU-mutagenised male and is hence inherited with the C57BL/6 alleles, was mapped to a 8.47 Mb region flanked by the SNPs rs28002552 and rs4223216 on chromosome 2C1.3–C2. This region contained 95 genes which included the <i>Galnt3</i> gene. (<b>B</b>) DNA sequence analysis of <i>Galnt3</i> identified a T to A transversion in codon 589, such that the wild type (WT) sequence, TGG which encodes an evolutionarily conserved tryptophan (Trp) residue was altered to the mutant (m) sequence, AGG which encodes an arginine (Arg) residue. (<b>C</b>) Amplification refractory mutation system (ARMS) PCR was used to confirm the presence of the mutation by designing primers (n, normal (WT) and m, mutant) that yielded 307 bp WT and 230 bp mutant PCR products, respectively. PCR amplification of <i>Gapdh</i> was used as a control for the presence of DNA. N = numbers of mice with each genotype. (D) Protein sequence alignment (CLUSTALW) of Galnt3 from 5 species revealed that the Trp (W) residue is evolutionarily conserved in the Galnt3 orthologues of mouse, human, monkey, xenopus and zebrafish.</p

Analysis of gene expression in bone and kidneys.

<p>RNA from femora and kidneys was extracted from WT littermates (black) (2 males and 1 female) and <i>Tcal/Tcal</i> (white) (2 males and 1 female) adult mice, aged 18–20 weeks. Quantitative reverse transcriptase-PCR (qRT-PCR) was used to study the expression of: (<b>A</b>) <i>Galnt3</i> and (<b>B</b>) <i>Fgf23</i> in femora; and (<b>C</b>) <i>Kl</i>, (<b>D</b>) <i>Cyp27b1</i>, (<b>E</b>) <i>Slc34a1</i>, and (<b>F</b>) <i>Slc34a3</i> in kidneys. Samples were analysed in triplicate (n = 3 mice for each group i.e. total of 9 samples) and mRNA levels were normalized to <i>Gapdh</i> and expressed as fold change (mean ± SEM) compared to WT. The data from males and females were combined, as differences in plasma biochemical analysis between the genders had not been observed (Fig. 5). The expression of <i>Galnt3</i> and <i>Fgf23</i> was significantly increased in the bone of <i>Tcal/Tcal</i> mice when compared to that of WT littermates; however, the expression of the renal expressed genes <i>Kl</i>, <i>Cyp27b1</i>, <i>Slc34a1</i> and <i>Slc34a3</i> was not significantly different in the <i>Tcal/Tcal</i> mice compared to WT littermates. P-values are from unpaired Students t-test (*p<0.05, **p<0.01).</p

Plasma biochemical analyses.

<p>Plasma from WT littermates (+/+) (black), <i>Tcal</i>/+ (grey) and <i>Tcal/Tcal</i> (white) adult mice, aged 10 weeks was obtained and used to measure plasma concentrations of (<b>A</b>) phosphate, (<b>B</b>) alkaline phosphatase (ALP) activity, (<b>C</b>) calcium (adjusted for albumin concentrations), (<b>D</b>) PTH, (<b>E</b>) intact Fgf23, and (<b>F</b>) 1,25-dihydroxyvitamin D. <i>Tcal/Tcal</i> mice but not <i>Tcal</i>/+ mice, had: hyperphosphataemia, reduced alkaline phosphatase activity, reduced plasma concentrations of intact Fgf23, and elevated circulating concentrations of 1,25-dihydroxyvitamin D. The plasma concentrations of calcium and PTH were not significantly different. Sufficient volumes of plasma samples from female mice were not available for an analysis of 1,25-dihydroxyvitamin D concentrations; however, analysis of pooled samples from 2 WT female littermates, 3 <i>Tcal</i>/+ females, and 3 <i>Tcal/Tcal</i> females revealed that the plasma 1,25-dihydroxyvitamin D concentrations were 58.2 pmol/l, 55.7 pmol/l, and 71.7 pmol/l, respectively. These results indicate that the plasma 1,25-dihydroxyvitamin D concentrations in WT littermates and <i>Tcal</i>/+ females are similar, but are elevated in <i>Tcal/Tcal</i> females, and are consistent with the observations in male mice. The data are represented as mean ± SEM, and p-values are from unpaired Students t-test (*p<0.05, **p<0.01, ***p<0.001).</p