Canine NAPEPLD-associated models of human myelin disorders

Abstract

Canine leukoencephalomyelopathy (LEMP) is a juvenile-onset neurodegenerative disorder of the CNS white matter currently described in Rottweiler and Leonberger dogs. Genome-wide association study (GWAS) allowed us to map LEMP in a Leonberger cohort to dog chromosome 18. Subsequent whole genome re-sequencing of a Leonberger case enabled the identification of a single private homozygous non-synonymous missense variant located in the highly conserved metallo-beta-lactamase domain of the N-acyl phosphatidylethanolamine phospholipase D (NAPEPLD) gene, encoding an enzyme of the endocannabinoid system. We then sequenced this gene in LEMP-affected Rottweilers and identified a different frameshift variant, which is predicted to replace the C-terminal metallo-beta-lactamase domain of the wild type protein. Haplotype analysis of SNP array genotypes revealed that the frameshift variant was present in diverse haplotypes in Rottweilers, and also in Great Danes, indicating an old origin of this second NAPEPLD variant. The identification of different NAPEPLD variants in dog breeds affected by leukoencephalopathies with heterogeneous pathological features, implicates the NAPEPLD enzyme as important in myelin homeostasis, and suggests a novel candidate gene for myelination disorders in people.

Figure 1

Positional cloning of the LEMP-associated locus in Leonbergers. (a) Manhattan plot for the GWAS using 14 LEMP-affected dogs and 186 control dogs is shown and indicates a signal with multiple associated SNPs on chromosome 18. The -log P-values for each SNP are plotted on the y-axis versus each canine autosome and the X chromosome on the x-axis. The red line represents the Bonferroni corrected significance threshold (−log (P) = 6.35). A mixed model analysis corrected for population stratification was carried out as described in the Methods. Inset: Corrected QQ-plot confirms that the actually observed P-values of the best associated markers have stronger association with the trait than expected by chance (null hypothesis, red line). (b) Haplotype analysis of SNP array genotypes of 14 cases and 28 carriers allowed fine mapping of the critical region for LEMP to a 0.9 Mb interval. Each line represents a unique haplotype. (c) The LEMP-associated region contains 14 loci including NAPEPLD gene.

Figure 2

The NAPEPLD missense variant detected in LEMP-affected Leonbergers. (a) Chromatograms of wild type, carrier, and an affected dog indicate the c.538 G > C variant which changes codon 180 (shown below). (b) The variant is located in exon 3 of canine NAPEPLD that encodes a functionally important domain of the NAPEPLD protein. (c) The predicted p.Ala180Pro exchange affects an evolutionary conserved residue. The multiple sequence amino acid alignment was done using accessions XP_005631036.1 (Canis lupus familiaris), NP_001116310.1 (Homo sapiens), NP_001015680.1 (Bos taurus), XP_014594420.1 (Equus caballus), NP_848843.1 (Mus musculus), NP_955413.1 (Rattus norwegicus), NP_001025901.1 (Gallus gallus), XP_002933136.1 (Xenopus tropicalis) and NP_001074082.2 (Danio rerio).

Figure 3

The NAPEPLD frameshift variant detected in LEMP-affected Rottweilers. (a) Chromatograms of wild type, carrier, and an affected dog indicate the c.345_346insC variant. (b,c) The schematic representation of the canine NAPEPLD gene indicates that the 1 bp insertion is located in exon 3 and leads to a frameshift which is predicted to produce a novel 186 amino acid long C-terminus of NAPEPLD and replaces the metallo-beta-lactamase domain of the wild type protein.

Figure 4

Across breed haplotype analysis in Rottweilers and Great Danes indicates an old mutation event. (a) Diverse haplotypes were detected exploring SNP array genotypes of four LEMP-affected Rottweilers (RO) and 23 heterozygous carriers of the NAPEPLD frameshift variant in Rottweilers and Great Danes (GD). Each line represents a unique haplotype. (b) A 50 kb-sized identical haplotype in all dogs contains segments of the canine NAPEPLD and ARMC10 genes.

Figure 5

Phenotypic variability of transverse spinal cord sections of LEMP-affected dogs. Combined luxol fast blue/hematoxylin & eosin stain of paraffin sections. (a) Thoracic spinal cord of a Leonberger case L1 with typical LEMP lesions as previously described. Note the bilateral-symmetrical loss of myelin in the corticospinal tracts as indicated by the loss of the blue color. (b) Cervical spinal cord of a previously described Rottweiler case R1 with similar lesions as in (a). This dog had severe white matter loss in some areas combined with axonal loss, infiltration by macrophages and capillary hypertrophy. (c) Thoracic spinal cord of Leonberger case L3. The quality of lesions is similar as in (a), but the distribution is different with lesions being most severe in the dorsal tracts. (d) Thoracic spinal cord of Rottweiler case R3 showing similar lesions, which are less defined and more widespread encroaching into lateroventral tracts.

Figure 6

Histopathology of the spinal cord of LEMP-affected dogs. Combined luxol fast blue/hematoxylin & eosin stain of paraffin sections. (a,b) Spinal cord sections of previously described Rottweiler case R2 showing deep blue staining of the normal myelin in an unaffected area (a) and an affected spinal cord area (b) exhibiting loss of blue myelin staining, vacuoles (asterisk), and large gemistocytic astrocytes (arrow). (c) Affected spinal cord area of the previously described Rottweiler case R1 exhibiting severe loss of blue myelin staining, vacuoles (asterisk), infiltration by macrophages (black arrowhead), capillary hypertrophy (brown arrowhead) and large gemistocytic astrocytes (arrow).