Mutations in the CYP27B1 gene cause vitamin D dependent rickets in pugs

Abstract

Rickets is a disorder of bone development and can be the result of either dietary or genetic causes. Here, related pugs from 2 litters were included. Three pugs had clinical signs including, lameness, bone deformities, and dyspnea. One other pug was found dead. Radiographs of 2 affected pugs, 5 and 6 months old, showed generalized widening, and irregular margination of the physes of both the appendicular and the axial skeleton with generalized decrease in bone opacity and bulbous swelling of the costochondral junctions. Two pugs had low serum calcium and 1,25 (OH)₂ D₃ concentrations. Test results further indicated secondary hyperparathyroidism with adequate concentrations of 25-hydroxyvitamin D. Necropsy revealed tongue-like projections of cartilage extending into the metaphysis consistent with rickets, loss of metaphyseal mineralization and lung pathology. Vitamin D-dependent rickets was diagnosed. A truncating mutation in the 1α-hydroxylase gene (CYP27B1) was identified by genome sequence analysis of the pugs with VDDR type 1A. Vitamin D-dependent rickets type 1A can occur in young pugs, and if left untreated is a life-threatening condition. Early medical intervention can reverse clinical signs and should be instituted as soon as possible.

Keywords: bone; hypocalcemia; pug; vitamin D.

FIGURE 1

Family pedigree of litter 1 and litter 2. Filled circles (female) or squares (male) indicate homozygous (TT) or dead subjects; circles and squares with dots indicate heterozygous subjects (GT); and empty circles and squares indicate unaffected (GG) (n = 2) or not genetically investigated (n = 3) subjects.

FIGURE 2

Radiographic examinations of the antebrachium and carpus (CrCd/DPa—projections) before and after initiation of corrective treatment with 1,25‐dihydroxyvitamin D3, (Etalpha). The physeal widening, irregular marginations of the metaphyses, and osteopenia improve over time. Radiographic changes are most dramatic in the distal radius and ulna. There is unbalanced growth between the radius and ulna resulting in elbow joint incongruity with secondary subchondral sclerosis. (A) Pug 2 with VDDR type 1A before treatment. (B) Pug 2 with VDDR type 1A after 6 weeks on treatment with 1,25‐dihydroxyvitamin D3 (Etalpha). (C) Age‐matched control pug (the dog has an unrelated defect in the elbow joint).

FIGURE 3

(A) Computed tomography images of the entire spine of Pug 1.1♀ with VDDR type 1A showing generalized decrease in bone density (attenuation). Endplate widening is also noted. (B) Computed tomography images of the entire spine of an age‐matched control pug.

FIGURE 4

(A) Histopathological changes at sites of enchondral ossification in the bones of Pug 1.1♀ with VDDR type 1A. Costochondral joint, longitudinal section stained with hematoxylin and eosin (H&E), showing retention of hypertrophic chondrocytes and tongue‐like projections of cartilage extending into the metaphysis from the physeal cartilage. (B) Histopathological changes at sites of enchondral ossification in the bones of Pug 1.1♀ with VDDR type 1A. Vertebrae T13–L1, transverse section (H&E), showing disorganized columns of hypertrophic chondrocytes and tongue‐like projections of cartilage in the metaphysis. (C) Spinal cord at T13–L1, transverse section (H&E), showing focal malacia with moderate parenchymal destruction and gliosis of primarily the ventral funiculi at the level of spinal cord stenosis.

FIGURE 5

A stop gain mutation (chr10:2182971G>T) in CYP27B1 identified from the 2 pugs (Pug 1.1 and Pug 2) with VDDR type 1A. This premature truncation occurs at the 87th codon in exon 2, which leads to 83% of the protein sequence to be missing.