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. 2011 Apr 15;6(4):e18931.
doi: 10.1371/journal.pone.0018931.

An Unusual Splice Defect in the Mitofusin 2 Gene (MFN2) Is Associated With Degenerative Axonopathy in Tyrolean Grey Cattle

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An Unusual Splice Defect in the Mitofusin 2 Gene (MFN2) Is Associated With Degenerative Axonopathy in Tyrolean Grey Cattle

Cord Drögemüller et al. PLoS One. .
Free PMC article


Tyrolean Grey cattle represent a local breed with a population size of ∼5000 registered cows. In 2003, a previously unknown neurological disorder was recognized in Tyrolean Grey cattle. The clinical signs of the disorder are similar to those of bovine progressive degenerative myeloencephalopathy (weaver syndrome) in Brown Swiss cattle but occur much earlier in life. The neuropathological investigation of an affected calf showed axonal degeneration in the central nervous system (CNS) and femoral nerve. The pedigrees of the affected calves suggested a monogenic autosomal recessive inheritance. We localized the responsible mutation to a 1.9 Mb interval on chromosome 16 by genome-wide association and haplotype mapping. The MFN2 gene located in this interval encodes mitofusin 2, a mitochondrial membrane protein. A heritable human axonal neuropathy, Charcot-Marie-Tooth disease-2A2 (CMT2A2), is caused by MFN2 mutations. Therefore, we considered MFN2 a positional and functional candidate gene and performed mutation analysis in affected and control Tyrolean Grey cattle. We did not find any non-synonymous variants. However, we identified a perfectly associated silent SNP in the coding region of exon 20 of the MFN2 gene. This SNP is located within a putative exonic splice enhancer (ESE) and the variant allele leads to partial retention of the entire intron 19 and a premature stop codon in the aberrant MFN2 transcript. Thus we have identified a highly unusual splicing defect, where an exonic single base exchange leads to the retention of the preceding intron. This splicing defect represents a potential explanation for the observed degenerative axonopathy. Marker assisted selection can now be used to eliminate degenerative axonopathy from Tyrolean Grey cattle.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.


Figure 1
Figure 1. Phenotype of degenerative axonopathy in Tyrolean Grey cattle.
(A) Moderate to severe spinal ataxia in an affected calf. Gait abnormalities and proprioceptive deficits were present in all limbs, but were markedly more pronounced in the hind than in the front limbs. (B) Thoracic spinal cord of a calf affected with degenerative axonopathy. Bilateral-symmetrical axonal loss leading to reduction of axonal density in the dorsal spinocerebellar tract (arrows) and gracile fascicle (arrowheads). Modified Bielschowsky staining, 12.5× magnification. (C) Swollen and chromatolytic neuron (arrow) adjacent to a normal neuron (arrowhead) in the mesencephalic red nucleus. The nucleus is displaced at the periphery and pyknotic. H&E, 400× magnification. (D) Femoral nerve. Wallerian type axonal degeneration and axonal loss: digestion chambers containing axonal and myelin fragments (arrows), proliferation of Schwann cells forming bands of Büngner. H&E, 400× magnification. (E, F, G) Corresponding tissue sections of a normal age-matched control animal.
Figure 2
Figure 2. Genome-wide association and homozygosity mapping of Tyrolean Grey cattle degenerative axonopathy.
(A) Case-control genome-wide association analysis shows a significant association to SNPs on chromosome 16. (B) Single SNP association results across BTA 16. (C) Homozygosity mapping of the degenerative axonopathy mutation. The analysis of SNP genotypes from 14 affected calves indicated an extended shared homozygous region of 2.9 Mb. One recombinant chromosome from a carrier animal further narrowed the critical interval to 1.9 Mb. (D) Gene content of the corresponding human chromosome 1 segment.
Figure 3
Figure 3. MFN2 transcript analysis.
(A) Northern blot using a full length MFN2 cDNA probe. Note the additional band of 5.8 kb in all examined tissues of an affected calf. (B) Relative MFN2 transcript levels. The MFN2 expression levels were normalized to BM2 expression. For the affected calf the relative expression levels of the wildtype and mutant transcript are indicated. (C) Bovine MFN2 gene organization. The 2229C-wildtype allele gives rise to a transcript, which is spliced as expected. The mutant 2229T-allele leads to partial retention of intron 19. Thus from the mutant allele tissue-specific varying fractions of wildtype transcript and an aberrant transcript encoding a truncated MFN2 protein are expressed.
Figure 4
Figure 4. Western immunoblot analysis of MFN2.
Extracts from the cerebral and cerebellar cortex of a degenerative axonopathy affected calf and a healthy control calf were analyzed. In the samples from the affected calf a weak additional MFN2 band of slightly lower molecular mass (83.7 kDa, arrow) as compared to the full-length wild-type protein (86.3 kDa) is visible. Conversely, in the healthy control only the wild-type MFN2 is detected. Molecular masses of the size standard are indicated on the right.
Figure 5
Figure 5. Electron micrographs of striated muscle fibers.
(A) Muscle fibers of healthy calf contain rigorously oriented, elongate mitochondria. (B) Uneven space between myofibrils in striated muscle of affected calf displays branched mitochondria. (scale bar  = 0.5 µm)

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