A Nonsense Variant in COL6A1 in Landseer Dogs with Muscular Dystrophy

Abstract

A novel canine muscular dystrophy in Landseer dogs was observed. We had access to five affected dogs from two litters. The clinical signs started at a few weeks of age, and the severe progressive muscle weakness led to euthanasia between 5 and 15 months of age. The pedigrees of the affected dogs suggested a monogenic autosomal-recessive inheritance of the trait. Linkage and homozygosity mapping indicated two potential genome segments for the causative variant on chromosomes 10 and 31 harboring a total of 4.8 Mb of DNA or 0.2% of the canine genome. Using the Illumina sequencing technology, we obtained a whole-genome sequence from one affected Landseer. Variants were called with respect to the dog reference genome and compared with the genetic variants of 170 control dogs from other breeds. The affected Landseer dog was homozygous for a single, private nonsynonymous variant in the critical intervals, a nonsense variant in the COL6A1 gene (Chr31:39,303,964G>T; COL6A1:c.289G>T; p.E97*). Genotypes at this variant showed perfect concordance with the muscular dystrophy phenotype in all five cases and more than 1000 control dogs. Variants in the human COL6A1 gene cause Bethlem myopathy or Ullrich congenital muscular dystrophy. We therefore conclude that the identified canine COL6A1 variant is most likely causative for the observed muscular dystrophy in Landseer dogs. On the basis of the nature of the genetic variant in Landseer dogs and their severe clinical phenotype these dogs represent a model for human Ullrich congenital muscular dystrophy.

Keywords: Mendelian traits; animal model; dog; veterinary genetics; whole-genome sequencing.

Figure 1

Skeletal muscle pathology. (A) M. quadriceps of a 4-months-old healthy Landseer dog with normal muscle texture without signs of significant pathologic changes. Especially, muscle and fiber size configuration and variation, sarcolemma, nuclear structure and distribution as well as endomysial connective tissue are of physiological appearance. hematoxylin and eosin (HE) staining, 100×. (B) Same muscle in the Gomori-Trichrome staining according to Engel. Normal muscle tissue and fiber structure without considerable pathologic changes are obvious, especially intrasarcoplasmal texture, mitochondria, muscular nuclei, and endomysial connective tissue are unobtrusive. (C) M. quadriceps of an affected full-sibling of the control dog shown above. HE staining, 100×. The muscle texture is severely disturbed showing a pathologic muscle fiber variation in size and configuration, signs of de- and regeneration, e.g., nuclear proliferation and distribution, as well as a remarkable increase and distortion of the endomysial connective tissue. (D) Same muscle in the Gomori-Trichrome staining according to Engel points up the pathologic muscle changes. Interestingly and in contrast to other forms of muscle dystrophy such as the Duchenne/Becker analogous disease in Golden Retrievers, the sarcoplasm structures and the mitochondria are not markedly altered. (E) Immunohistochemistry with an antibody against dystrophin performed on the same muscle as in (C) and (D). The diseased muscle shows no deficiency and regular expression of dystrophin. Thus this particular muscular dystrophy is markedly different from the Duchenne/Becker types of muscular dystrophy, where dystrophin expression is altered.

Figure 2

Mapping strategy. (A) A family comprising six offspring with their parents and one isolated case were available for the initial mapping of the disease locus. We performed parametric linkage analysis for a recessive trait in the family and homozygosity analysis across the four cases. (B) The analyses yielded five linked genome segments (orange) and 18 homozygous genome segments (red). Only two regions on chromosomes 10 and 31 showed both linkage and homozygosity and were considered the critical intervals (arrows). Specifically, these regions corresponded to Chr10:61,871,450-66,047,210 and Chr31:38,752,158-39,364,930.

Figure 3

Electropherograms of the COL6A1:c.289G>T variant. A fragment harboring exon 3 and flanking sequences of the COL6A1 gene was amplified by polymerase chain reaction and sequenced with the Sanger method. Shown are representative traces from Landseer dogs with the three different genotypes. The position of the variant is indicated by an arrow.