Figure 1.
Morphological features of the affected Brazilian Terriers.
Deformed legs with crooked radiocarpal joints and prominent joint hyperlaxity especially in the hind limbs (A) and a brachycephalic craniofacial morphology (C) presented by a 4-week-old Brazilian Terrier puppy compared to a healthy littermate (B,D). The affected puppies (n = 3) were severely growth retarded and weighted 35% less than their healthy littermates (n = 4) at the age of three weeks (E).
Figure 2.
Radiographic and histological features of skeletal disease in Brazilian Terrier puppies.
Comparison of the hind limbs of an affected 4-week-old puppy (A) and a healthy littermate (B) revealed epiphyseal dysplasia in the affected dog. The ossification centers of the epiphyses of the proximal tibia (long arrow) and tarsal bones (short arrow) are smaller in the affected puppy while the epiphyses of the healthy littermate are normally mineralized. The vertebral endplates of the lumbar spine (arrow) are poorly mineralized in the affected (C) puppy as compared with the healthy littermate (D). The skull of an affected 6-week-old Brazilian Terrier (E) has brachycephalic appearance with shortened maxilla. Radiopacity of the bones is reduced. The cervical vertebral endplates are not mineralized (arrow). A skull of a normal littermate is shown in comparison (F). Histological examination of the skeletal structures of a 4-week-old affected puppy showed normal growth plate with resting, proliferating and hypertrophic layers of chondrocytes in the tibia (G) but lack of secondary ossification center and occasional separate spots of loose fibrous tissue without sign of osteogenesis (I). The lack of secondary ossification centers and irregularities of the growth plate were present in the vertebral bodies (J). The epiphysis of a 5-week old healthy puppy is shown in comparison (H). Scale bar 100 µm.
Figure 3.
An example of our large Brazilian Terrier pedigree established around the affected puppies.
The pedigree with multiple affected dogs in the litters, equal gender distribution and healthy parents suggests a recessive mode of inheritance. Squares note males and circles females. Black-filling indicates affected dogs. Genotypes of the GUSB gene (T/T = homozygous mutation allele, C/C = homozygous wild type allele) are shown below the boxes. Dogs used in the NGS are marked by asterisks.
Figure 4.
A genome-wide association study reveals an associated locus at CFA6.
Manhattan plot indicates the best SNP with a raw and an empirical p-value after 100 000 permutations (A). Affected dogs share a 13-Mb homozygous haplotype, which contains over 200 genes (B). Colors in haplotype block are as follows: plum marks the homozygous allele, orange heterozygous allele and yellow an opposite homozygous allele. Dogs used for the NGS are marked by asterisks. A targeted sequencing of the entire locus identified a homozygous c.866C>T mutation in the exon 5 of the GUSB gene (C). The mutation was found by next generation sequencing (D) and was validated by Sanger sequencing (E).
Table 1.
SNP and indel statistics of the NGS of the identified locus in Brazilian Terriers.
Figure legend.
The conservation of the GUSB mutation site.
A multiple alignment of the substitution site (P289) indicates a high conservation across taxa including eukaryotic and prokaryotic species. The p.P289L mutation is predicted to be pathogenic and likely disrupts the structure of the GUSB enzyme.
Table 2.
Lysosomal enzyme activities in the serum of affected and healthy Brazilian Terriers.