A homozygous missense variant in the alkaline phosphatase gene ALPL is associated with a severe form of canine hypophosphatasia

Abstract

Inherited skeletal disorders affect both humans and animals. In the current study, we have performed series of clinical, pathological and genetic examinations to characterize a previously unreported skeletal disease in the Karelian Bear Dog (KBD) breed. The disease was recognized in seven KBD puppies with a variable presentation of skeletal hypomineralization, growth retardation, seizures and movement difficulties. Exome sequencing of one affected dog revealed a homozygous missense variant (c.1301T > G; p.V434G) in the tissue non-specific alkaline phosphatase gene, ALPL. The identified recessive variant showed full segregation with the disease in a cohort of 509 KBDs with a carrier frequency of 0.17 and was absent from 303 dogs from control breeds. In humans, recessive and dominant ALPL mutations cause hypophosphatasia (HPP), a metabolic bone disease with highly heterogeneous clinical manifestations, ranging from lethal perinatal hypomineralization to a relatively mild dental disease. Our study reports the first naturally occurring HPP in animals, resembling the human infantile form. The canine HPP model may serve as a preclinical model while a genetic test will assist in breeding programs.

Figure 1

Radiographic findings in a 3-month-old affected dog. (a) The oldest affected dog (litter 1) had a crouched, planti- and palmigrade stance and gait. (b) Laterolateral radiograph of the head. Both maxilla and mandible are shortened and the lower jaw is malformed with an angulated mandibular body and an elongated mandibular ramus (arrowhead). (c) Laterolateral radiograph of the lumbar spine. The vertebral bodies are short, the end plates are poorly mineralized and the dorsal borders of the vertebral spinous processes are irregular. Mediolateral radiograph of (d) the right front and (e) right hind limbs. The long bones are hourglass-shaped (open arrowheads) and have hypoplastic marrow cavity, as well as hypoplastic cortical bone. Carpal bones and distal femoral epiphyses show irregular mineralization (arrowheads).

Figure 2

Pathological findings in canine hypophosphatasia. (a) A cross section of a formalin fixed femur from a 3-month-old affected puppy (litter 1). The femur has a striking hourglass appearance. The diaphyseal periosteum and cortex are abnormally broadened by unmineralized osteoid and fibrous tissue, and the marrow cavity is narrowed due to defective modelling of primary spongiosa (encircled). Retained epiphyseal cartilage (open arrowhead) and irregular growth lines (arrowhead) are seen within the epiphyseal areas. (b) Formalin-fixed cerebellum of a 2-week-old affected puppy (litter 2). The cerebellar vermis is protruding into the spinal canal (arrowhead). (c) Longitudinal section of diaphyseal cortex, decalcified, HE 50X. The periosteal cambium (C) is thickened and cellular, with an underlying broad zone of unmineralized woven bone (W) and a cortex consisting of lamellar bone (L) instead of compact bone. The periosteal fibrous layer (F) appears normal. (d) Articular cartilage and epiphysis, decalcified, HE 50X. The ossification front is uneven (line) and irregular tongues of retained poorly mineralized cartilage (arrowheads) are present within the epiphysis. (e) Thyroid gland from an affected puppy, HE 200X. The C-cells are numerous and hypertrophic between the colloid follicles. (f) Thyroid gland from an affected puppy, calcitonin IHC 200X. Profound C-cell hypertrophy and hyperplasia is noted. (g) Thyroid gland of an unaffected, age- and gender matched puppy, calcitonin IHC 200X. A few C-cell groups and scattered single calcitonin-rich C-cells are seen. All histopathological images (c–g) are from the 3-month-old affected puppy (litter 1).

Figure 3

A disease pedigree constructed around the affected dogs. The ALPL genotypes are denoted with different colours. Several inbreeding loops and common ancestors are present. Within the affected litters, samples were obtained from altogether three parents and twelve unaffected full siblings. In both litters 1 and 2, one puppy had died without sampling or clinical and pathological examinations, at the age of 3 weeks (litter 1) and 2 days (litter 2). The genotype and possible affection status of these puppies could not therefore be determined.

Figure 4

A missense change in the canine ALPL gene. (a) Sequence chromatograms of the ALPL c.1301T > G variant in exon 11 and a schematic representation of the ALPL gene. (b) Structure of the TNSALP polypeptide and multiple sequence alignment of the variant position in 18 mammalian species. Amino acid residues that make up the active site and the homodimerization interface are scattered along the polypeptide chain, whereas the calcium binding site and the crown domain form separate entities. The p. V434G missense change is located within the crown domain. Those amino acid residues that contain HPP-associated variants in humans are marked on top of the alignment and highlighted in grey (http://www.sesep.uvsq.fr/03_hypo_mutations.php). (c) Side and top views of the three-dimensional (3D) structure of the TNSALP homodimer, modelled from the crystal structure of the human placental alkaline phosphatase. The two monomers are separated by different colours. The Val⁴³⁴ position is denoted with yellow, the active site residues with red and the collagen binding motif with black. The crown domain forms a flexible loop structure on top of the homodimer.