Spontaneous asj-2J mutant mouse as a model for generalized arterial calcification of infancy: a large deletion/insertion mutation in the Enpp1 gene.

Generalized arterial calcification of infancy (GACI), an autosomal recessive disorder caused by mutations in the ENPP1 gene, manifests with extensive mineralization of the cardiovascular system. The affected individuals in most cases die within the first year of life, and there is currently no effective treatment for this disorder. In this study, we characterized a spontaneous mutant mouse, asj-2J, as a model for GACI. These mice were identified as part of a phenotypic deviant search in a large-scale production colony of BALB/cJ mice at The Jackson Laboratory. They demonstrated a characteristic gait due to stiffening of the joints, with phenotypic similarity to a previously characterized asj ("ages with stiffened joints") mouse, caused by a missense mutation in the Enpp1 gene. Complementation testing indicated that asj-2J and asj were allelic. PCR-based mutation detection strategy revealed in asj-2J mice a large, 40,035 bp, deletion spanning from intron 1 to the 3'-untranslated region of the Enpp1 gene, coupled with a 74 bp insertion. This was accompanied with a significant reduction in the plasma PPi concentration and reduced PPi/Pi ratio. As a consequence, extensive aberrant mineralization affecting the arterial vasculature, a number of internal organs, and the dermal sheath of vibrissae, a progressive biomarker of the ectopic mineralization process, was demonstrated by a combination of micro computed tomography, histopathology with calcium-specific stains, and direct chemical assay of calcium. Comparison of the asj and asj-2J mice demonstrated that the latter ones, particularly when placed on an acceleration diet high in phosphate and low in magnesium, had more extensive mineralization. Thus, the asj-2J mouse serves as a novel model for GACI, a currently intractable disorder

Skin fragility in the wild-derived, inbred mouse strain Mus pahari/EiJ.

Mus pahari is a wild-derived, inbred mouse strain. M. pahari colony managers observed fragility of this strain\u27s skin resulting in separation of tail skin from the mouse if handled incorrectly. Tail skin tension testing of M. pahari resulted in significantly lowered force threshold for caudal skin rupture and loss in comparison to closely related inbred mouse species and subspecies and even more than a model for junctional epidermolysis bullosa. Histologically, the tail skin separated at the subdermal level with the dermis firmly attached to the epidermis, excluding the epidermolysis bullosa complex of diseases. The dermal collagen bundles were abnormally thickened and branched. Elastin fiber deposition was focally altered in the dermis adjacent to the hair follicle. Collagens present in the skin could not be differentiated between the species in protein gels following digestion with pepsin. Together these data suggest that M. pahari have altered extracellular matrix development resulting in separation of the skin below the level of the dermis with moderate force similar to the African spiny mouse (Acomys spp.). Exp Mol Pathol 2017 Feb; 102(1):128-132

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

A missense mutation in Grm6 reduces but does not eliminate mGluR6 expression or rod depolarizing bipolar cell function.

GRM6 encodes the metabotropic glutamate receptor 6 (mGluR6) used by retinal depolarizing bipolar cells (DBCs). Mutations in GRM6 lead to DBC dysfunction and underlie the human condition autosomal recessive complete congenital stationary night blindness. Mouse mutants for Grm6 are important models for this condition. Here we report a new Grm6 mutant, identified in an electroretinogram (ERG) screen of mice maintained at The Jackson Laboratory. The Grm6(nob8) mouse has a reduced-amplitude b-wave component of the ERG, which reflects light-evoked DBC activity. Sequencing identified a missense mutation that converts a highly conserved methionine within the ligand binding domain to leucine (p.Met66Leu). Consistent with prior studies of Grm6 mutant mice, the laminar size and structure in the Grm6(nob8) retina were comparable to control. The Grm6(nob8) phenotype is distinguished from other Grm6 mutants that carry a null allele by a reduced but not absent ERG b-wave, decreased but present expression of mGluR6 at DBC dendritic tips, and mislocalization of mGluR6 to DBC somas. Consistent with a reduced but not absent b-wave, there were a subset of retinal ganglion cells whose responses to light onset have times to peak within the range of those in control retinas. These data indicate that the p.Met66Leu mutant mGluR6 is trafficked less than control. However, the mGluR6 that is localized to the DBC dendritic tips is able to initiate DBC signal transduction. The Grm6(nob8) mouse extends the Grm6 allelic series and will be useful for elucidating the role of mGluR6 in DBC signal transduction and in human disease.NEW & NOTEWORTHY This article describes a mouse model of the human disease complete congenital stationary night blindness in which the mutation reduces but does not eliminate GRM6 expression and bipolar cell function, a distinct phenotype from that seen in other Grm6 mouse models. J Neurophysiol 2017 Aug; 118(2):845-854