Identification of the Identical Human Mutation in ACVR1 in 2 Cats With Fibrodysplasia Ossificans Progressiva

Abstract

Two domestic shorthair cats, 1 intact female and 1 intact male, presented with progressive limb lameness and digital deformities at 4 and 6 months of age. Stiffness and swelling of the distal thoracic and pelvic limb joints progressed to involve hip and shoulder joints, resulting in reduced mobility. Radiographs in both cats and computed tomography of the male cat revealed ankylosing, polyarticular deposits of extracortical heterotopic bone spanning multiple axial and appendicular joints, extending into adjacent musculotendinous tissues. All findings supported fibrodysplasia ossificans progressiva (FOP), a disorder characterized by toe malformations and progressive heterotopic ossification in humans. In both cats, molecular analyses revealed the same heterozygous mutation in the activin A receptor type I (ACVR1) gene that occurs in humans with FOP. Several reports of heterotopic ossification in cats exist, but this is the first one to identify clinical FOP in 2 cats with the identical mutation that occurs in >95% of humans with FOP.

A 6½-month-old, intact-male, American domestic short hair (DSH) cat (case No. 1) presented to the referring veterinarian with a history of progressive pelvic limb lameness. Physical examination initially suggested elongated third digits in all feet, relative to other digits (Fig. 1, front limb), and radiographs revealed malformations with excessive mineralization affecting the third metatarsi and phalanges (Fig. 2, hindlimb before surgery). Assumed to be the cause of pelvic limb lameness, both hind third digits were amputated at the metatarsophalangeal joint. Around the same time in New Zealand, a 4-month-old intact-female DSH cat (case No. 2) presented with lameness, similar digit malformations on all limbs, and a firm interdigital swelling on its right forelimb. The mass was firmly attached to the third metacarpal and was removed surgically. Despite the appearance that there was elongation of the third digit, measurements revealed that the ratio of the length (measured from the proximal end of the metacarpal bone to the distal end of the intermediate phalanx) of the second digit to that of the third digit was significantly decreased (P < .01) in both cats when compared to normal cats (81.9% ± 3.0% and 90.2% ± 2.0% of normal, respectively). The same measurement comparing the third to the fourth digit revealed that the third digit was longer than the fourth digit by 13% to 30% in affected animals when compared to normal cats, in which the third and fourth digits are approximately equal in length.

Figures 1–2.

Feline fibrodysplasia ossificans progressiva (FOP), distal pelvic limbs, cat, case No. 1. Right pelvic limb, 6.5 months of age. There is shortening of the second digit with relative elongation of the third digit (arrow). Figures 2. Left hindlimb, wild-type (WT) cat on left and FOP cat on right. Digits are labeled (2–5; there is no first digit on the pelvic limbs in most cats). There is relative elongation and thickening of the third digit (which should normally be the same length and thickness as the fourth digit) and shortening and thickening of the second digit (which should be the same length and width as the fifth digit). The third metatarsophalangeal and the proximal and distal inter-phalangeal joints have aberrant periarticular mineralized exostoses with bridging ankylosis. Feline fibrodysplasia ossificans progressiva, cat, case No. 1.

Following surgery, stiffness progressed dramatically in both cats, affecting all 4 limbs within 2 and 4 weeks, respectively. Loss of muscle became evident in the hindlimbs and progressed cranially over the dorsum, sparing the neck. Euthanasia was elected for case No. 2 at 5 months of age due to rapidly declining quality of life. Its limbs and radiographic images were sent to Massey University, New Zealand. Case No. 1 was referred to the University of Pennsylvania with a tentative diagnosis of fibrodysplasia ossificans progressiva (FOP).⁹ Stiffness continued to progress involving coxofemoral joints, spine, and thoracic limbs. By 1.5 years of age, the cat was non-ambulatory with stiffened pelvic limbs joints, kyphosis, loss of voluntary motion, and conscious proprioceptive deficits in both pelvic legs, suggesting radiculoneuropathy. The cat continued to be bright and alert, but the quality of life steadily declined, necessitating humane euthanasia at 19 months of age. Both cats were adopted as strays; thus, no family history was available.

Radiographs and computed tomography images obtained from case No. 1 at 14 and 19 months of age demonstrated extensive smooth extraskeletal bone present primarily along the dorsal aspect of the vertebrae, spanning the dorsal spinous processes and fusing the dorsal articular facets. Irregular, well-defined extraskeletal bone was present along the cranial aspect of the left coxofemoral joint, which blended with a large mass of superimposed bone densities surrounding both coxofemoral joints and extending craniodorsally along the iliac wings of the pelvis (Suppl. Fig. S1). Smooth extensions of ectopic bone arose from the caudal aspects of the distal femurs and extended to the caudal aspects of the proximal tibiae. Extraskeletal bone was present along the caudal aspects of the distal tibiae, extending to the dorsal surfaces of each calcaneus and spanning the caudal aspects of the tibiotarsal joints (Suppl. Figs. S1, S2). Cortices of the caudodistal humeral diaphyses were thickened, leading to large, irregular bone formations spanning the caudal aspects of the elbow joints, fusing the distal humeri and the dorsal, non-articular surfaces of the olecranon processes (Suppl. Figs. S1, S3). In case No. 2, complete pelvic limb and distal thoracic limb radiographs were available. These revealed multiple partly mineralized tissue densities surrounding or adjacent to carpal joints and several digits, often associated with a periosteal reaction. Large, partly mineralized masses bridged the caudal aspect of both stifle joints and merged with the cortical surfaces of the distal femur and proximal tibia on both hindlimbs. The polyostotic periarticular bone formation, representing ossification of ligaments, tendons, and muscle, was consistent with the diagnosis of FOP⁹ in both cats.

Gross postmortem examination identified musculoskeletal leions that corresponded to the radiographic findings, including bony ankylosis of multiple joints due to large deposits of periarticular extracortical ectopic bone (bony exostoses). Multiple appendicular joints in all 4 limbs were severely affected (Fig. 3 and Suppl. Fig. S4), as well as the caudal thoracic and lumbar vertebrae (Fig. 4). Skull and cervical vertebrae were minimally affected (not shown). As the excessive bony exostoses obscured the anatomic landmarks and prevented further gross dissection of joints and surrounding soft tissues, select unilateral segments of the appendicular and axial skeleton were boiled to better reveal the extent of the extraskeletal bone (Figs. 3, 4 and Suppl. Fig. S4), while nonboiled contralateral segments were sectioned with a bandsaw and decalcified in an aerated solution of 10% formic acid, changed every 2 days, until bones were soft enough to be sectioned with a microtome blade for routine preparation of histologic sections (Figs. 5–7).

Figure 3–4.

Figure 3. Pelvic limb. Corresponding to the radiographic images (Suppl. Figs. S1–S3), there is ossification of the musculotendinous origin of the left gastrocnemius muscle (arrowhead). Exostoses bridge and fuse the right tibiotarsal joint at about 90 degrees of flexion and ossify the musculotendinous insertion of the gastrocnemius muscle (arrow). Figure 4. Caudal thoracic and lumbar spine. T13–L2 vertebral segments—upper image: lateral view, T13 at the right, dorsal spinous processes are at the top of the image. Lower image: ventral view, T13 at the right, vertebral bodies are at the top of the image. There is extensive heterotopic ossification that completely bridges the dorsolateral aspects of the vertebrae and partially bridges the ventrolateral aspects. L3–S3 vertebral segment—upper image: lateral view, L3 is at the right; lower image: dorsal view, L3 is at the right, dorsal spinous processes are at the top of the image. There is extensive smooth heterotopic ossification that completely bridges the dorsal aspects of the L3–S1 segments. A large, unilateral nodular exostosis extends from the right dorsolateral surface of L7–S1 (arrowheads). The caudal sacral segment (S3) remains unfused to the cranial segments (arrow), a normal finding in a juvenile-adult cat. Feline fibrodysplasia ossificans progressiva, cat, case No. 1.

Figure 5–7.

Figure 5. Femorotibial joint, sagittal section. There is articular cartilage degeneration, including erosions and ulcers (arrows) with hypereosinophilic cartilage matrix. A membrane of fibroadipose tissue (asterisk) extends from the periphery across the thinned articular cartilage. Figure 6. Left distal humerus. A partially ossified, cartilaginous exostosis (arrows) is present beyond the profile of the metaphyseal cortex (CTX), within the surrounding adipose tissue. Growth cartilage of metaphyseal growth plate (P) persists but is capped by a transverse seam of bone, indicating growth cessation. Figure 7. Left distal humerus. The exostosis has central area of hyaline cartilage (CE) that merges into islands of woven bone with the development of medullary spaces (asterisks) that demonstrate active remodeling with resorption lacunae containing multinucleate osteoclasts around the periphery (arrows). The exostosis extends into and partially effaces periarticular ligamentous tissue (L).

Histologically, the left coxofemoral, right femorotibial, left tibiotarsal, and right elbow joints (both cases) and phalanges (case No. 2) had similar lesions. Articular cartilage had mild to advanced degenerative changes characterized by multifocal erosions and ulcers partially covered by fibroadipose (ie, pannus) tissue extending from the periphery toward the center of the joint surface (Fig. 5). The cartilage had hypereosinophilic matrix and fibrillation with empty lacunae (chondrocyte necrosis), paucicellular foci (chondrocyte loss), and scattered clusters of chondrocyte lacunae (chondrones). Metaphyseal physes were retained, and nodular exostoses of partially ossified bone extended radially from the physes, often well beyond the profile of the cortex (Fig. 6). Nodular periarticular exostoses had active endochondral ossification with formation of medullary spaces containing adipose tissue, vascular sinuses, and occasionally hematopoietic tissue (Fig. 7) and often extended into and effaced the adjacent ligaments, tendons, and muscle (Fig. 3 and Suppl. Fig. S4). Thoracic and abdominal viscera as well as endocrine organs were within normal limits (not shown).

Because of the clinical similarities (digit malformations and extensive extraskeletal endochondral ossification) to human FOP (MIM #135100; http://omim.org/entry/135100), a condition caused by activating mutations in the ACVR1 gene¹⁵ (also known as ALK2), we designed feline-specific polymerase chain reaction (PCR) primers for ACVR1 genomic DNA to amplify all exons and splice junctions (Suppl. Table S1). ACVR1 encodes a bone morphogenic protein type I receptor that has been highly conserved through vertebrate evolution (Suppl. Fig. S5). Genomic DNA was isolated from the blood of the FOP cats and a control DSH, and ACVR1 PCR products were sequenced. Both FOP cats were heterozygous at complementary DNA (cDNA) position c.617 in ACVR1, carrying 1 wild-type (c.617G) and 1 mutated allele (c.617A) (Suppl. Fig. S5). This mutation is the same single-nucleotide substitution that occurs in most cases of humans with “classic” FOP and in the knock-in mouse model of FOP³ and causes an amino acid substitution from arginine to histidine at codon 206 (R206 H).^6,15

Fibrodysplasia ossificans progressiva in cats (also called progressive ossifying myositis and myositis ossificans) was described in the literature as early as 1980; since then, 12 additional case reports have been published, most recently in 2013^{1,2,5,11,13,16–20} (Suppl. Table S2). Unrelated males and females were affected, including domestic shorthair and long-hair cats as well as 1 Maine Coon and 1 Himalayan cat, suggesting no sex or breed predilection. For most affected cats, including both cases reported here, no family history or DNA samples from relatives were available. However, known family histories^2,19 support that spontaneous, dominant new mutations cause FOP in those cats. In the knock-in mouse model of FOP, autosomal dominant inheritance has been shown, with the c.617G>A nucleotide substitution alone sufficient to induce all aspects of the phenotypes observed in human FOP.³ These findings are consistent with human FOP, which shows no racial, ethnic, sex, or geographic bias. When inheritance in families occurs, transmission is autosomal dominant.^6,8

Clinical signs in all affected cats reported in the literature mirrored those seen in the cats reported here, with the characteristic increasing stiffness and loss of mobility. In some cats, the stiffness began in either the front limbs or in the pelvic limbs. In the absence of injury, heterotopic ossification in human FOP characteristically begins in the upper back and neck and then progresses distally.⁸ In both of our cases, surgical amputation of the toes could be responsible for the enhanced heterotopic ossification in the hindlimbs relative to the forelimbs. Consistent with human and murine disease, in most cats, clinical signs were progressive, as was the formation of heterotopic bone.

In murine and human FOP, heterotopic bone formation is typically present in skeletal muscle, ligaments, and tendons. The early stages of bone-forming lesions are associated with the presence of immune cells and tissue destruction and turnover that is followed by an anabolic stage of tissue replacement through cartilage and bone formation.^4,6,8 As in our cats with FOP, affected mice and humans develop abnormal bone formation at joints that causes joint fusions; degenerative joint disease and osteochondromas are also common.^3,6

Weightbearing joints were the most prominent sites of ectopic bone formation in both cats. The degenerative changes in articular surfaces were most likely secondary to the development of aberrant forces and malalignment generated from partial to complete ankylosing ossification of periarticular structures. The muscle atrophy in affected cats may be secondary due to disuse or, given the presence of vertebral exostoses and ankylosis, compressive radiculoneuropathies. Unlike humans but similar to FOP mice, neck muscles in both cats were minimally affected. Plausibly, the mild lesions at this site may result from differences in posture and workload between cats, mice, and humans.

Both cats presented in this report showed some impaired movement prior to surgery as reported by the owner. However, the rate of disease progression greatly accelerated post-surgery, consistent with a response to trauma (such as intramuscular injections, surgery to remove extra bone) in humans and mice.^4,7,8,12,14 A diagnostic feature of murine and human FOP are congenital malformations of the great toe (first digit); malformed thumbs are observed less frequently and usually less severe.^3,6 In our feline FOP cases, primarily the second digits were stunted. Interestingly, the digital malformations in our patients were not noted in cats from previous reports, raising the possibility that these cats carried a different mutation resulting in a milder phenotype¹⁰ or perhaps a different genetic defect.

This report describes the clinical signs, radiographic features, and pathological features in 2 cats that are strikingly similar to those in mice and humans with FOP. Commonalities include episodes of extensive heterotopic endochondral ossification that typically begin or progress after inflammation or trauma, malformed toes, osteochondromas, and the presence of the c.617G>A mutation in the ACVR1 gene.