A one base pair deletion in the canine ATP13A2 gene causes exon skipping and late-onset neuronal ceroid lipofuscinosis in the Tibetan terrier

Abstract

Neuronal ceroid lipofuscinosis (NCL) is a progressive neurodegenerative disease characterized by brain and retinal atrophy and the intracellular accumulation of autofluorescent lysosomal storage bodies resembling lipofuscin in neurons and other cells. Tibetan terriers show a late-onset lethal form of NCL manifesting first visible signs at 5-7 years of age. Genome-wide association analyses for 12 Tibetan-terrier-NCL-cases and 7 Tibetan-terrier controls using the 127K canine Affymetrix SNP chip and mixed model analysis mapped NCL to dog chromosome (CFA) 2 at 83.71-84.72 Mb. Multipoint linkage and association analyses in 376 Tibetan terriers confirmed this genomic region on CFA2. A mutation analysis for 14 positional candidate genes in two NCL-cases and one control revealed a strongly associated single nucleotide polymorphism (SNP) in the MAPK PM20/PM21 gene and a perfectly with NCL associated single base pair deletion (c.1620delG) within exon 16 of the ATP13A2 gene. The c.1620delG mutation in ATP13A2 causes skipping of exon 16 presumably due to a broken exonic splicing enhancer motif. As a result of this mutation, ATP13A2 lacks 69 amino acids. All known 24 NCL cases were homozygous for this deletion and all obligate 35 NCL-carriers were heterozygous. In a sample of 144 dogs from eleven other breeds, the c.1620delG mutation could not be found. Knowledge of the causative mutation for late-onset NCL in Tibetan terrier allows genetic testing of these dogs to avoid matings of carrier animals. ATP13A2 mutations have been described in familial Parkinson syndrome (PARK9). Tibetan terriers with these mutations provide a valuable model for a PARK9-linked disease and possibly for manganese toxicity in synucleinopathies.

Figure 1. Luxol fast blue (LFB) staining.

Of the (A) cerebrum and (B) retina from a NCL-affected Tibetan terrier. Intracytoplasmic LFB positive pigment is indicated with arrows, bar indicates magnification for the micrograph (50 µm).

Figure 2. Mutation analysis of exon 16 using cDNA.

Primer pairs used were ATP13A2_F7 and ATP13A2_R7. Agarose gel analyses revealed exon 16 skipping for NCL-affected Tibetan terriers.

Figure 3. Detection of the ATP13A2 one base pair deletion.

The deletion was genotyped using a restriction fragment length polymorphism on a 4% agarosegel and the restriction enzyme BglI. NCL-affected Tibetan terriers show only one fragment, NCL-carriers show two fragments and NCL-unaffected Tibetan terriers show only one fragment, because the 312 and 323 bp fragments are not separated on the agarosegel.