Non-invasive demonstration of ectopic mineralization in <i>asj-2J</i> mice by micro CT.

<p>Note the extensive mineralization in the muzzle skin containing the dermal sheath of vibrissae and in the ears (A, arrows); in the juxta-articular connective tissue in the elbows (B, arrows); axial view of dorsal artery (C, arrow); spinal and intercostal tissues (D, arrows); and in the right rib fusion leading to scoliosis as well as in the ears (E, arrows).</p

Calcium, phosphorus and pyrophosphate concentrations in serum/plasma of mice maintained on a regular rodent diet.

<p>Blood samples were collected by cardiac puncture, Ca and P concentrations were determined in serum, and PP_i levels were measured in heparinized plasma after removal of platelets. Statistical significance in comparison to <i>Enpp1^+/+</i> mice: *<i>P</i><0.01.</p><p>Calcium, phosphorus and pyrophosphate concentrations in serum/plasma of mice maintained on a regular rodent diet.</p

Aberrant tissue mineralization in <i>Enpp1^+/+</i>, <i>Enpp1^+/asj-2J</i> and <i>Enpp1^asj-2J</i> mice on normal and acceleration diet compared with <i>Enpp1^asj</i> mice on acceleration diet.

<p>Mice were placed on either normal diet or acceleration diet at 4 weeks of age, and tissues were collected for histopathology at 3 months of age. The values represent the number of mice, as percent of all animals examined, affected by mineralization as examined by hematoxylin and eosin stain on one section. Statistical analyses were performed with Fisher’s Exact test. Comparison to <i>Enpp1^asj-2J</i> mice on normal diet: *<i>P</i><0.05; **<i>P</i><0.01. Comparison to <i>Enpp1^asj-2J</i> mice on acceleration diet: ⁺<i>P</i><0.05.</p><p>Aberrant tissue mineralization in <i>Enpp1^+/+</i>, <i>Enpp1^+/asj-2J</i> and <i>Enpp1^asj-2J</i> mice on normal and acceleration diet compared with <i>Enpp1^asj</i> mice on acceleration diet.</p

Enhanced mineralization in <i>asj-2J</i> mice placed on “acceleration diet”, rich in phosphate and low in magnesium.

<p>The mice were placed on this diet at 4 weeks of age and necropsy was performed at 12 weeks. Note extensive mineralization as visualized by special stain (Alizarin Red). Assessment of the histopathology suggested that the mineralization was more extensive in mice kept on acceleration diet compared to the same mice kept on control diet (compare mineralization in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113542#pone-0113542-g001" target="_blank">Fig. 1B</a>).</p

Schematic presentation of the large deletion/insertion mutation in the <i>Enpp1</i> gene in <i>asj-2J</i> mice, and PCR-based genotyping.

<p><u>A:</u> Note the 40,035 bp deletion extending from intron 1 to the 3′-untranslated region of the gene. The deleted segment is replaced by insertion of a 74 bp fragment which is derived from the 3′ UTR of the gene. <u>B:</u> Development of specific primers (p1/p2/p3), with positions shown in A, allowed genotyping of the wild-type (WT) and mutant alleles and identification of wild-type, heterozygous and mutant homozygous mice. nc: negative control without DNA.</p

Phenotypic presentation and extensive ectopic mineralization of <i>asj-2J</i> mice.

<p><u>A:</u> Note the appearance of stiffened front and hind feet in <i>asj-2J</i> mice (arrows, lower panel) in comparison to a wild-type littermate. <u>B:</u> Note the extensive mineralization (red color) in the dermal sheath of vibrissae, arterial blood vessels, and various internal organs in <i>asj-2J</i> mice, as visualized by special stain (Alizarin Red).</p

Quantitation of ectopic mineralization by chemical assay of calcium content in the muzzle skin (A), aorta (B), carotid artery (C), and kidney (D) in 12-week old <i>asj-2J</i> mice kept either on normal mouse diet or placed on acceleration diet at 4 weeks of age.

<p>The statistical analysis was performed with Kruskal-Wallis nonparametric test; *, p<0.01 in comparison to wild-type <i>Enpp1^+/+</i> mice; ⁺p<0.01 as compared with <i>asj-2J</i> mice kept on regular diet. Values are mean ± SE, n = 6–12 per group.</p