Homozygous CNP Mutation and Neurodegeneration in Weimaraners: Myelin Abnormalities and Accumulation of Lipofuscin-like Inclusions

Abstract

A progressive neurological disorder was observed in a male neutered Weimaraner. Clinical signs included fecal incontinence, lethargy, moderate paraparesis, proprioceptive pelvic limb ataxia, falling, cognitive decline, incoordination, decreased interest in food, changes in posture, and episodes of trance-like behavior. Neurologic signs were first observed at approximately 4 years, 10 months of age and progressed slowly. Magnetic resonance imaging showed generalized brain atrophy with areas of white matter pathology. Humane euthanasia was elected at 6 years, 7 months of age due to increasing severity of the neurological signs. Autofluorescent intracellular granules were observed in the cerebral and cerebellar cortexes, optic nerve, and cardiac muscle of the affected dog. These abnormal inclusions in the cerebral cortex and cardiac muscle immunolabeled with antibodies to mitochondrial ATP synthase subunit c protein, like that observed in the neuronal ceroid lipofuscinosis group of lysosomal storage diseases. Immunolabeling also demonstrated pronounced neuroinflammation in brain tissues. The ultrastructural appearances of the disease-related inclusion bodies in the brain and optic nerve were quite variable. The ultrastructure and locations of many of the inclusions in the nervous tissues suggested that they were derived, at least in part, from the myelin surrounding axons. The storage bodies in the cardiac muscle were located in mitochondria-rich regions and consisted of parallel arrays of membrane-like components interspersed with electron-dense flocculent material. The disease was characterized by pronounced abnormalities in the myelin of the brain and optic nerve consisting of distinctive areas of ballooning between the layers of myelin. The whole genome sequence generated from the affected dog contained a homozygous G-to-A missense mutation in CNP, which encodes proteins with CNPase enzyme activity and a structural role in myelin. The mutation predicts a Thr42Met amino acid sequence substitution. Genotyping of archived Weimaraner DNA samples identified an additional G > A variant homozygote with a clinical history and brain lesions similar to those of the proband. Of 304 Weimaraners and over 4000 other dogs of various breeds, the proband and the other Weimaraner that exhibited similar signs were the only two that were homozygous for the CNP missense variant. CNPase immunolabeling was widespread in brain tissues from normal dogs but was undetectable in the same tissues from the proband. Based on the clinical history, fluorescence and electron-microscopy, immunohistochemistry, and molecular genetic findings, the late-onset Weimaraner disorder likely results from the missense mutation that results in CNPase deficiency, leading to myelin abnormalities, accumulation of lysosomal storage bodies, and brain atrophy. Similar disorders have been associated with different CNP variants in Dalmatians and in human subjects.

Neuronal Ceroid Lipofuscinosis in a Mixed-Breed Dog with a Splice Site Variant in CLN6

“…The SRA BioSample identifier for the proband is SAMN39309507. The SRA BioSample identifiers of the remaining whole genome sequences used in this analysis were reported previously [ 6 ]. The amino acid positions for canine CLN6 were numbered according to XP_038298694.1 (NCBI).…”

“…In addition to the 13 genes that have been associated with NCLs by consensus among many researchers, other hereditary disorders in dogs with NCL-like phenotypes have been described that result from mutations in genes that have not been associated with NCL in human subjects. Among these genes are ARSG and CNP [ 5 , 6 , 7 ]. For dogs that exhibit the characteristic signs of NCL, a whole genome sequence (WGS) analysis can be used to determine whether the disorder is associated with a mutation in any of these genes.…”

Neuronal Ceroid Lipofuscinosis in a Mixed Breed Dog with a Splice Site Variant in CLN6

“…In addition to the 13 genes that have been associated with NCLs by consensus among many researchers, other hereditary disorders in dogs with NCL-like phenotypes have been described that result from mutations in genes that have not been associated with NCL in human subjects. Among these genes are ARSG and CNP [5][6][7]. For dogs that exhibit the characteristic signs of NCL, whole genome sequence (WGS) analysis can be used to determine whether the disorder 2 is associated with a mutation in any of these genes.…”

Nanobodies against the myelin enzyme CNPase as tools for structural and functional studies

“…The molecular function of CNPase is not fully understood, and a high-resolution structure of full-length CNPase has not been experimentally solved. The catalytic function of the N-terminal polynucleotide kinase-like domain is controversial, while the reaction catalysed by the C-terminal phosphodiesterase catalytic domain has been known in vitro for >60 years [15] and characterised mechanistically in detail [16–18]. However, it is unclear if the physiological function of CNPase is related to its enzymatic properties, or if it has evolved to be more relevant through its molecular interactions with other proteins [19, 20], RNA [16, 21, 22], and lipid membranes [23, 24].…”

“…Deficiency of CNPase in mice causes ultrastructural defects of the axon/myelin-unit [8, 9] and impairs the initiation of executive functions [10, 11]. A homozygous missense mutation in the human gene encoding CNPase correlates with white matter loss and associated neurodegeneration [12], and CNP gene mutations in dogs have been linked to lysosomal storage disease and myelin abnormalities [13, 14]. Due to its high abundance in myelinating glia, CNPase is a commonly used marker of myelin, being localised in the non-compacted subcompartments.…”