Molecular characterization of two canine models of human leukoencephalopathies uncover NAPEPLD as new candidate gene

Canine leukoencephalomyelopathy (LEMP) is a recessively inherited juvenile-onset neurodegenerative disorder of the CNS white matter. LEMP is characterized by slowly worsening gait abnormalities, especially spontaneous knuckling, dragging of the paws and hypermetria (over-shooting) of the thoracic limbs, and a characteristic pattern on cervical MRI. Affected dogs show corresponding gross lesions in the cervical spinal cord white matter that may extend to the thoracic spinal cord, as well as to the brain; peripheral nerve and muscle biopsies are unremarkable. LEMP has been described in Rottweilers and Leonbergers, and there is a striking similarity of the lesions in these two breeds.We utilized Leonberger LEMP cases (n=14) diagnosed via magnetic resonance imaging, necropsy, and/or neurologist assessment, and neurologically healthy Leonberger controls over the age of 8 (n=139), to map LEMP to CFA 18 by GWAS (Pcorrected = 3.7 x10-20). Haplotype analysis of LEMP-affected Leonbergers showed a 3.3 Mb area of extended shared homozygosity from 16.6 to 19.9 Mb on CFA 18. Whole genome re-sequencing of a Leonberger LEMP case enabled the identification of a private homozygous non-synonymous variant located in exon 2 of the N-acyl phosphatidylethanolamine phospholipase D (NAPEPLD) gene. All LEMP-affected Leonbergers were homozygous for the NAPEPLD missense mutation; the mutant allele frequency in a global population of 4,588 Leonbergers was 0.08. All 4 exons of the NAPEPLD gene were Sanger sequenced in 9 LEMP affected Rottweilers (variously confirmed by characteristic MRI changes, neurologist assessment, and/or necropsy). All were clear of the Leonberger mutation, but a new frameshift mutation in exon 2 was discovered in which 4 of 9 LEMP affected Rottweilers were homozygous, another case was heterozygous, and 4 other cases were homozygous for the reference allele. The NAPELPD frameshift allele frequency in a population of 144 Rottweilers was 0.05. Subsequent haplotype analysis of SNP array genotypes revealed that the frameshift mutation was present in two diverse haplotypes in affected Rottweilers. The NAPEPLD protein is a phospholipase D type enzyme that catalyzes the release of N-acylethanolamine from N-acyl-phosphatidylethanolamine, and in so doing generates N-arachidonoylethanolamine, the ligand of cannabinoid and vanilloid receptors. There is as yet no association known for involvement of this gene in demyelinating disorders in people. These results provide evidence for allelic and locus heterogeneity in canine LEMP, an old origin of the allele in Rottweilers, and a novel description of defects in the endocannabinoid system associated with severe neurodegenerative disease.