Fig 1.
Some paternal and maternal individuals were repetitively used for breeding, resulting in an increase in genetic homogeneity.
Fig 2.
Clinical and pathological findings of NAD in Papillon dogs.
a) Case 1. The dog showed persistent intention tremor and was severely emaciated due to eating difficulty. The appendicular muscles were atrophied. b) Photographic example of the formalin-fixed brain of Case 1. The cerebellum shows mild atrophy (yellow arrowheads). c) Sagittal section of the cerebellum of Case 2. The cerebellar cortex is mildly atrophied and the sulci are dilated (arrowheads). HE stain. d) Numerous small to medium-sized spheroids (yellow arrowheads) are observed in the cerebellar granular layer. Vacuolization is also observed at the border of the granular layer and white matter (black arrowheads). HE-stain. Bar = 50 μm. e) The olivary nucleus of Case 1. Small to medium-sized spheroids are scattered around the neurons. LFB-HE stain. Bar = 50 μm. f). The cuneate nucleus of Case 1. Medium-sized to large spheroids are frequently observed. The spheroids are eosinophilic and granular, and some contain clefts and vacuoles in the core. LFB-HE stain. Bar = 50 μm. g) to i) The cuneate nucleus of Case 1. Bar = 50 μm. NF (g) and synaptophysin (h) immunohistochemistry showed strong reactivity in the axonal spheroids. i) Ubiquitin immunohistochemistry showed numerous granularly positive spheroids. The surrounding small spheroids are also strongly positive. j) Transmission electron microscopy of a spheroid in the dorsal horn of the spinal cord. The spheroid contains dense membranous material and swollen mitochondria. Bar = 2 μm.
Table 1.
Number of SNPs and indel mutations identified through the WES analysis and candidate selection.
Table 2.
Overview of all variants identified by exome sequencing and in silico pathogenicity analysis.
Table 3.
Results of TaqMan genotyping assay for the PLA2G6 c.1579G>A mutation in Papillon, Chihuahua and other breeds.
Fig 3.
Structures of the PLA2G6 transcript and iPLA2β protein.
a) Structure of the canine PLA2G6 gene. The PLA2G6 gene contains 16 exons with a total length of 49.03 kb. The identified mutation in this study, c.1579G>A, was located at exon 10. b) Protein structure of human and canine iPLA2β and the homology of the ankyrin-repeat domain, patatin domain, and two calmodulin-binding domains. ANK; ankyrin domain Calmod; calmodulin domain and GTSTG; lipase motif. Yellow and blue bands in human iPLA2β indicate known substitutions and deletion mutations in human INAD, respectively. The yellow band in the canine iPLA2β indicates the T526A mutation identified in this study. c) ClustalW multiple protein alignment of the region of iPLA2β containing the mutation. The target alanine is shown in the red box. Sequences for dogs (E2RPF9_CANFA), humans (PLPL9_HUMAN), mice (PLPL9_MOUSE), rats (PLPL9_RAT), cows (E1B889_BOVIN), horses (F6WG51_HORSE), cats (F6WG51_FELCA), rabbits (G1SQE9_RABIT), pigs (F1SKP4_PIG), chickens (F1NBV7_CHICK), African clawed frogs (Q6DDK0_XENLA) and zebrafish (Q6NWY0_DANRE) are shown. The target alanine lies within a region of 13 highly conserved amino acids that contains the GTSTG lipase domain.
Fig 4.
iPLA2β immunohistochemistry of Case 1 (a, c) and a normal (b, d) canine brain.
(a) Spheroids in the cerebellum showed faint granular expression (black arrowheads). Cytoplasmic granular expression was also observed in the Purkinje cells (white arrowheads). (b) The expression was very weak in the cerebellum of the control dog. In the brainstem (c, d), intense cytoplasmic and nuclear expression was detected in the neurons of both the NAD and control dogs. Axonal spheroids in the neuropil of the brainstem of Case 1 showed an intense granular expression (arrowheads). Bar = 50 μm.