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Abstract

Congenital skeletal abnormalities may be genetic, teratogenic, or nutritional in origin; distinguishing among these different causes is essential in the management of the disease but may be challenging. In some cases, teratogenic or nutritional causes of skeletal abnormalities may appear very similar to genetic causes. For example, chondrodysplasia associated with intrauterine zinc or manganese deficiency and mild forms of hereditary chondrodysplasia have very similar clinical features and histologic lesions. Therefore, historical data are essential in any attempt to distinguish genetic and acquired causes of skeletal lesions; as many animals as possible should be examined; and samples should be collected for future analysis, such as genetic testing. Acquired causes of defects often show substantial variation in presentation and may improve with time, while genetic causes frequently have a consistent presentation. If a disease is determined to be of genetic origin, a number of approaches may be used to detect mutations, each with advantages and disadvantages. These approaches include sequencing candidate genes, single-nucleotide polymorphism array with genomewide association studies, and exome or whole genome sequencing. Despite advances in technology and increased cost-effectiveness of these techniques, a good clinical history and description of the pathology and a reliable diagnosis are still key components of any investigation.

Keywords

genetic disease bone diseases skeletal abnormalities nutrition genomewide association study sheep cattle pig diagnostic investigation

Skeletal abnormalities are commonly encountered in farm animals; in one study of congenital abnormalities in lambs in Australia, 52% of abnormalities involved the skeletal system.²⁹ The abnormalities may be sporadic or occur as outbreaks, and they may be genetic or teratogenic in origin or secondary to intrauterine nutritional deficiency. Pathologists must be careful not to suggest a genetic basis without good reason: this can have important consequences for the farmer and stud breeder, and it has the potential to result in litigation.

There are a large number of potential genetic skeletal abnormalities in humans; 456 syndromes, organized into 40 groups, have been described on the basis of clinical features, radiographic abnormalities, and molecular defects.¹⁰⁹ As of 2011, mutations had been found in 226 genes to explain 316 diseases in humans.¹⁰⁹ Genetic skeletal diseases of animals are not described and classified in such detail; however, with the increasingly widespread availability of screening techniques, such as single-nucleotide microarrays and whole genome sequencing, the recognition of disease-causing mutations in animals has been revolutionized. The OMIA (Online Mendelian Inheritance of Animals) currently lists disease-causing mutations in 99 of 199 genetic disorders of cattle, 44 of 97 disorders of sheep, and 26 of 54 disorders of pigs.⁷⁴ Genetic skeletal diseases with known mutations and the proposed mechanisms are listed in Table 1, with descriptions and illustrations of a number of these recently reviewed.^55,101 A number of genetic mutations, such as developmental duplication in Angus cattle have not been published, as they have become the basis of patented commercial tests.¹³ Unfortunately, this leads to a delay in reporting of the gene associated with the defect and limits our ability to establish mechanisms behind these diseases, complete bone development pathways, and advance scientific knowledge.

Table 1.

Inherited Skeletal Diseases of Livestock With Known Mutations, OMIA Numbers,⁷⁴ and Proposed Mechanisms.

Disease	Breed / Mutation / OMIA No.	Proposed Mechanism
Bulldog chondrodysplasia ²¹
Breed	Dexter cattle, other miniature cattle breeds	ACAN is the main proteoglycan expressed by chondrocytes during cartilage formation in the primordial limb bud.
Mutated gene,	Aggrecan (ACAN), OMIA: 001271-9913
Angus dolichocephalic long-nosed dwarfism ⁶⁰
Breed	Angus cattle	PRKG2 is required for growth plate development as it regulates SOX9, a critical transcription factor involved in endochondral ossification and the control of growth plate collagens type II and X.
Mutated gene;	cGMP-dependent type II protein kinase (PRKG2); OMIA: 001485-9913
Ellis van Creveld syndrome 2 ^73,98
Breed	Japanese Brown cattle, Grey Alpine cattle	Part of the primary cilia, thought to have a role in regulating sonic hedgehog, a key regulator of skeletal development.
Mutated gene,	Ellis van Creveld syndrome 2 gene (EVC2), also known as LIMBIN; OMIA: 000187-9913
Spider lamb syndrome ¹⁴
Breed	Suffolk sheep, Hampshire sheep	Mutation removes FGFR3 inhibition of chondrocytes entering the hypertrophic chondrocyte phase, resulting in increased length of long bones.
Mutated gene,	Fibroblast growth factor receptor 3; OMIA: 001703-9940
Texel chondrodysplasia ¹¹⁵
Breed	Texel sheep	In addition to changes in the cartilage matrix, undersulfation of cartilage proteoglycans leads to altered Indian hedgehog signaling, resulting in decreased chondrocyte proliferation.²⁵
Mutated gene,	Sodium-sulfate transporter (SLC13A1); OMIA: 001400-9940
Schmid metaphyseal chondrodysplasia ⁷⁵
Breed	Yorkshire pig	Type X collagen is expressed by hypertrophic chondrocytes, and this mutation prevents trimerization of the collagen X chains.
Mutated gene,	Collagen, type X, alpha 1 chain (COL10A1); OMIA: 001718-9825
Arachnomelia ^17,37
Breed	Italian Brown, Simmental, German Fleckvieh and Brown Swiss cattle	Increased sulfite levels resulting in atypical bone development.
Mutated gene,	Molybdenum cofactor synthesis step 1 (MOCS1), sulfite oxidase (SUOX); OMIA: 001541-9913, 000059-9913
Osteopetrosis with gingival hamartomas ⁹³
Breed	Belgian blue cattle	CLCN7 and associated subunit osteopetrosis associated transmembrane protein (Ostm1) are on lysosome membranes and the ruffled border of osteoclasts. Mutations in CLCN7 and Ostm1 impair acidification of resorption lacunae
Mutated gene,	Chloride/proton exchanger lysosomal anion transporter (CLCN7); OMIA: 001887-9913
Osteopetrosis ⁷⁰
Breed	Red Angus cattle	The SLC4A2 transporter exchanges bicarbonate ions for chloride ions. Due to proton secretion during acidification of resorption lacunae, mutations in this transporter result in accumulation of bicarbonate ions, leading to toxic osteoclast alkalinisation.⁹²
Mutated gene,	Anion exchange transporter (SLC4A2); OMIA: 000755-9913
Marfan syndrome ⁵⁰
Breed	Japanese Black cattle	Fibrillin is a component of the extracellular microfibrils present in connective tissues. Fibrillins regulate TGF-β and BMP availability, and the decreased bone mineral density and decreased mechanical strength seen in Marfan syndrome are thought to be due to increased activation of TGF-β signaling.^9,76
Mutated gene,	Fibrillin 1 (FBN1); OMIA: 000628-9913
Autosomal recessive hypophosphatemic rickets, type 1 ¹¹⁴
Breed	Corriedale sheep	Dentin matrix protein 1 (DMP1) is a noncollagenous bone protein involved in bone mineralization. In addition, decreased DMP1 results in increased FGF23 and subsequent phosphaturia.⁹⁰
Mutated gene,	Dentin matrix protein 1 (DMP1); OMIA: 001542-9940
Vitamin D-dependent rickets type 1 ²³
Breed	Hannover pig	CYP27B1 is required for the formation of active vitamin D (1,25-dihydroxyvitamin D). Decreased active vitamin D leads to hypocalcemia and hypophosphatemia, impaired cartilage and bone mineralization, and decreased chondrocyte apoptosis.³¹
Mutated gene,	25-hydroxyvitamin D 1α-hydroxylase (CYP27B1); OMIA: 000837-9825
Inherited vitamin C deficiency ⁴⁶
Breed	od/od pig (Danish pig)	Mutation in GULO results in vitamin C deficiency and decreased collagen cross-linking.
Mutated gene,	L-gulonolactone oxidase (GULO)
Complex vertebral malformation ¹⁰⁴
Breed	Holstein cattle	SLC35A3 is a nucleotide sugar transporter, and mutations may result in abnormal Notch signaling. The Notch and Wnt pathways are essential for development of the axial skeleton.
Mutated gene,	UDP-N-acetylglucosamine transporter (SLC35A3); OMIA: 001340-9913
Brachyspina ²²
Breed	Holstein-Friesian cattle	The FANC proteins are involved in DNA cross-link repair. In zebrafish embryos, FANCI is highly expressed in the apical ectodermal ridge, an important area for fin/limb development.¹⁰⁵
Mutated gene,	Fanconi anemia, complementation group I (FANCI); OMIA: 000151-9913
Syndactyly ^36,38,54
Breed	Holstein-Friesian, Simmental cattle	LRP4 is involved in limb bud development, where it is expressed in the apical ectodermal ridge. It is also a negative regulator of LRP6-mediated activation of canonical Wnt signaling.⁶⁷
Mutated gene,	Lipoprotein receptor related protein 4/multiple epidermal growth factor 7 (LRP4/Mefg7); OMIA: 000963-9913
Tibial hemimelia ^13,62,79
Breed	Galloway, Shorthorn, Maine-Anjou	ALX4 is a negative regulator of sonic hedgehog and is involved in limb bud development. Mutations in this gene associated with polydactyly in mouse models and craniofacial defects in humans.^52,109,113
Mutated gene,	Aristaless-like homeobox-4 (ALX4); OMIA: 001009-9913

This review outlines an approach to investigating skeletal diseases in farm animals, with emphasis placed on investigating genetic causes.

Clinical History

A thorough clinical history is often key to distinguish between abnormalities due to genetic and nongenetic causes. A good example lies in investigating cases of chondrodysplastic dwarfism in cattle. A number of hereditary forms of chondrodysplasia with varying levels of severity are recognized, including relatively mild forms—such as “snorter” dwarfism in Herefords,⁵⁶ long-nosed dolichocephalic dwarfism in the Angus breed,⁶⁰ Ellis van Creveld 2 syndrome in Japanese Brown,⁷¹ and Grey Alpine breeds⁷³—and the severe form, Dexter bulldog dwarfism.⁴⁵ Mild forms of hereditary chondrodysplasia may be clinically and histologically indistinguishable from the disproportionate dwarfism associated with nutritional deficiencies of manganese or zinc (Figs. 1, 2). The mild genetic forms and the nutritional forms are both associated with a mild-moderate shortening of long bones and a slight decrease in thickness of the hypertrophic zone of the physis. It is seldom possible on the basis of clinical features alone to be confident of the etiology, but information such as pedigree details, knowledge of the number of animals affected, and maternal diet during pregnancy may provide useful clues.

Figures 1–4.
Chondrodysplasia. Figure 1. Grey Alpine calf with Ellis van Creveld syndrome showing disproportionate dwarfism and twisted limbs. Image courtesy of A. Gentile and C. Benazzi, University of Bologna, Italy. Figure 2. Calf with manganese deficiency showing disproportionate dwarfism and twisted limbs. Note the similarity in clinical presentation between calves in Figures 1 and 2. Figure 3. Dexter calf; note the domed head, severely stunted limbs and ventral abdominal hernia. Figure 4. Texel lamb showing disproportionate dwarfism and wide-based stance.

In general, a high incidence of skeletal disease in a cohort of newborn animals suggests a non-genetic etiology, unless the disease is caused by a new, dominantly inherited mutation in germ cell lines of a newly introduced sire, as occurs in severe forms of osteogenesis imperfecta.¹⁰ Recessively inherited diseases will only affect 1 in 4 offspring of a heterozygous dam mated to a heterozygous sire, and unless the defective gene is widespread throughout a herd or flock, the incidence is typically low. Even if 1 or more heterozygous sires are used excessively and mated to their offspring, an incidence close to 25% is highly unlikely. If the defect is due to recessive inheritance, then the affected animals are likely to be the offspring of the same sire or a closely related animal mated to related dams. In such cases, examination of pedigrees will be of considerable value.

If the skeletal abnormality has simple autosomal dominant inheritance, then evidence of disease might be expected in approximately 50% of the progeny of similarly affected sires or dams. However, some dominantly inherited skeletal diseases of animals are characterized by incomplete penetrance or codominance, and the abnormality is only partly expressed in heterozygous individuals. Spider lamb syndrome in Suffolk and Hampshire sheep⁹⁵ and bulldog chondrodysplasia in Dexter cattle²¹ are well-recognized examples of codominance in farm animals. In spider lamb syndrome, heterozygous animals are slightly taller than those not carrying the defective gene, while homozygous mutant animals express the characteristic lesions of the disease. In bulldog chondrodysplasia of Dexter cattle (Fig. 3), heterozygotes have short stature and are considered desirable by many lifestyle farmers, but homozygous animals with 2 copies of the defective gene are nonviable.

Skeletal deformities have been reported in cattle subjected to drought conditions in early pregnancy and fed an inappropriate diet, such as apple pulp,¹⁰⁷ or forced to eat potentially teratogenic plants that would otherwise be avoided or constitute an insignificant percentage of the ration.¹¹¹ In such cases, the percentage of newborn animals affected may be very high but the lesions highly variable due to exposure to the nutritional deficiency or teratogenic agent at different times during pregnancy. Insults during embryogenesis (first trimester) are likely to induce more significant defects. If the disorder is due to a teratogenic agent or intrauterine deficiency of a nutrient, such as manganese, mildly affected animals may recover following exposure to a normal ration after birth. This would not be expected if the cause were of genetic origin.

Infection of naïve dams with teratogenic viruses during pregnancy may result in outbreaks of congenital abnormalities on several properties in a region, depending on the distribution of their insect vectors, as seen regularly in parts of Eastern Australia with Akabane virus⁵⁹ and more recently with Schmallenberg virus in Europe.¹ However, when the virus is endemic in a region, then only occasional affected animals may be produced. The lesions associated with intrauterine exposure to Schmallenberg virus and other potentially teratogenic viruses, such as Akabane virus, bluetongue virus, and bovine viral diarrhea virus, typically involve multiple organ systems but can resemble skeletal diseases of genetic origin, such as complex vertebral malformation. In cases where a skeletal abnormality is confined to an individual animal, the cause is usually impossible to determine unless the lesions are consistent with a recognized genetic defect in a particular breed and the defective gene can be identified.

Examination of Affected Animals

It is important to examine as many affected animals as possible to accurately characterize the lesions and assess their variability. During autopsy, bones of endochondral and intramembranous origin should be examined with attention to rapidly growing physes, such as costochondral junctions, distal radius, proximal humerus, distal femur, and proximal tibia. Bone fragility should also be assessed, as increased fragility is a feature of certain inherited skeletal diseases, such as osteogenesis imperfecta and osteopetrosis.

Radiography and other forms of imaging may also provide valuable information and can be used either antemortem or postmortem to assess such features as bone shape, cortical thickness, and mineral density or to locate pathologic fractures.

Not all skeletal abnormalities will be associated with an abnormality in bone shape; therefore, sagittal sectioning of representative bones is recommended. In a recent investigation of gingival hamartomas in Belgian blue cattle, it was not until a mutation was found in a gene associated with osteopetrosis that the bones were examined and found to be osteopetrotic.⁹³

Although skeletal abnormalities of nutritional or toxic origin tend to be variable in presentation, inherited skeletal defects are typically more consistent. An exception to this paradigm is chondrodysplasia of Texel sheep (Fig. 4), where severely affected animals succumb to dyspnea and sudden death as a result of tracheal collapse, while mildly affected animals survive to adulthood and may go on to breed.¹⁰³

The trace element status of affected and control animals may provide useful information, but it is important to be aware that test results at the time of birth may not reflect those present several weeks or months previously when the skeleton was at an earlier stage of development. Liver samples from affected animals could be collected for manganese or zinc analysis if the animals have a short stature or for copper analysis if the bones are fragile.

If a genetic etiology is suspected, then whole blood, liver, ear notch, or tail hair samples for DNA extraction should be collected from all affected animals and from the dam, sire, and unaffected siblings (if there are any) to maximize the opportunity to detect a new mutation. We are often contacted about a suspected genetic abnormality near the end of a lambing/calving season, by which time there may be only 1 or 2 affected animals available and no samples have been collected from any others. In such cases, the chances of gaining enough information to distinguish between an acquired and a genetic abnormality or identifying a new genetic defect are remote.

Table 2 lists a number of differential diagnoses for increased bone fragility; Table 3 lists differential diagnoses for short stature; and Table 4 contains other possible presenting signs. Further details are included on the features of each disease that may help distinguish them.

Table 2.
Differential Diagnoses of Increased Bone Fragility in Livestock.

Differential Diagnoses Clinical Characteristics and Pathology

Osteogenesis imperfecta^6,10,51 May occur as “outbreaks” due to new dominant mutations in germline cells of sire; not breed associated Recessive inheritance in Barbados blackbelly sheep Often born dead or die within first few weeks of life Tissues affected: bones, tendons, eyes, teeth, ± skin Blue sclera, pink teeth, tendon and ligament hyperlaxity, intrauterine bone fractures, ± skin fragility in sheep Bones usually normal in shape Thin layer of abnormally basophilic osteoid lining cartilage spicules, lack of compaction of osteons in a cortex that consists of basophilic woven bone, trabecular microfractures

Osteopetrosis^{15,43,65,77,93} Recessive inheritance Breeds: Angus (black and red), Simmental, Belgian Blue, and Hereford cattle; Polypay sheep May be born premature and smaller in size Often born dead or die within first few weeks of life Bones less fragile than in osteogenesis imperfecta May have a domed head, brachygnathia inferior, and impacted molars Cones of persistent primary spongiosa extending into the diaphysis Osteoclasts may be absent or abundant, depending on the underlying genetic defect

Intrauterine copper deficiency^42,97,102 May be associated with other manifestations of copper deficiency in older animals (eg, swayback) Liver copper concentrations not necessarily decreased at the time of examination; bone formed during a period of copper deficiency will remain fragile even after copper concentrations have returned to normal Bones in lambs, calves, and pigs may be osteopenic with narrow cortices and decreased deposition of osteoid on cartilage spicules, resembling the scorbutic lattice of vitamin C deficiency In calves and pigs, physeal thickening due to enlargement of the hypertrophic zone and the presence of trabecular microfractures in the primary spongiosa may resemble rickets

Inherited rickets^32,40,66 Autosomal recessive in Corriedale sheep; autosomal dominant or X-linked inheritance reported in humans Hypocalcemia and hypophosphatemia Depending on the type of genetic defect 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D may be decreased, normal or increased Thickened physes in long bones, enlarged costochondral junctions (rachitic rosary) Increased thickness of hypertrophic zone in physes of rapidly growing bones, trabecular microfractures, disorganization of the primary spongiosa, poorly mineralized seams of osteoid lining trabeculae and filling Haversian systems in cortices

Inherited scurvy⁵³ Autosomal recessive in Danish od/od pig Decreased plasma ascorbic acid concentrations Fragile bones, subperiosteal and joint hemorrhages, petechiae, and bruising of the skin Sparse osteoblasts, osteoid absent from cartilage spicules in the primary spongiosa (scorbutic lattice) Cortices thin, physes narrow

Arachnomelia^18,100 Simmental, German Fleckvieh, Italian, and Swiss brown cattle Brachygnathia inferior with a turned up maxilla Long bone cortices normal in thickness but diaphyses abnormally thin Severe hyperextension of fetlocks, with kyphosis and scoliosis

Table 3.
Differential Diagnoses of Short Stature in Livestock.

Differential Diagnoses Clinical Characteristics and Pathology

Chondrodysplasia: genetic^{3,45,60,71,73} Usually has recessive inheritance, so only small numbers of animals affected Breeds: Angus, Dexter, Japanese Brown, Tyrolean Grey, and Holstein/Friesian cattle; Texel, Merino, and Cabugi sheep; Danish Landrace pigs Clinical signs have a consistent presentation typically with minimal variation Normal liver zinc and manganese concentrations Shortened long bones (mild to severe depending on the gene affected), epiphyses potentially mushroomed, domed head, ± brachygnathia inferior Decreased thickness of physes, particularly hypertrophic zone; histologic lesions present in severe forms but mild or nonexistent in other forms

Chondrodysplasia: nutritional^34,44,49,107 Prevalence generally higher than expected for inherited forms of chondrodysplasia May be a history of drought or adverse weather events during pregnancy and exposure to pregnant animals to unusual supplementary feeding or toxic plants Variation in severity of dwarfism and twisted limbs; animals with mild cases improve after birth Shortened long bones, epiphyses may be mushroomed Decreased thickness of physes, particularly hypertrophic zone; histologically lesions may be mild or nonexistent Low liver zinc or manganese concentrations; if deficiency occurred for a finite period during gestation, zinc or manganese concentrations may have returned to normal

Plant toxins^{16,58,63,82,110} Veratrum californicum toxicity in sheep: shortening metacarpal/metatarsal ± other bones, fusion metacarpal bones, arthrogryposis, hypermobility hock joints Wild lupins (Lupinus spp): cattle; shortening and rotation of limb bones, flexion contracture, arthrogryposis

Vitamin A toxicity^{20,33,69,89,112} Pigs: shortening of long bones, abnormally shaped long bones and carpal/tarsal bones, osteoporosis Calves: possibly associated with “hyena disease”; premature closure of hind limb growth plates, segmental narrowing of physes

Table 4.
Differential Diagnoses of Skeletal Abnormalities in Livestock.

Differential Diagnoses Clinical Characteristics and Pathology

Abnormal head shape

Chondrodysplasia See Table 3

Osteopetrosis⁸⁰ See Table 2; markedly thickened skull bones that in some animals may contain cystic cavities, impacted molars

Craniosynostoses²⁷ Possibly autosomal dominant with incomplete penetrance in Cabugi sheep; domed head, protruding mandible, exophthalmos, dwarfism

Toxic plants^{57,85,88,91,110} Veratrum californicum: sheep, cattle; cyclopia, harelip/cleft palate, mandibular micrognathia, tracheal/laryngeal stenosis Wild lupins (Lupinus spp): cattle; cleft palate, brachygnathia superior Prunus serotina (wild black cherry): pigs; cleft palate, arthrogryposis Datura stramonium: pigs; cleft palate, arthrogryposis, although definitive evidence lacking Leucaena, Mimosa: cattle/sheep; cleft palate, brachygnathia, agnathia, harelip, cranioschisis, forelimb flexure, scoliosis/kyphosis/torticollis

Vitamin A deficiency¹⁰¹ Cattle/pigs; failure of cranial cavity nerve foramina and spinal canal to enlarge

Vertebral abnormalities

Brachyspina^7,8,99 Cattle; smaller than normal size, shortened vertebral column due to fusion of vertebrae, and long thin limbs Irregular areas of ossification separated by cartilage, fusion of epiphyses, and diaphysis of adjacent vertebrae

Complex vertebral malformation^2,4,5 Cattle; smaller than normal size, consistent arthrogryposis of forelimbs ± hind limbs, shortening of cervical and thoracic vertebral column, hemivertebrae, fused vertebrae, scoliosis Heart defects in approximately 50% of affected animals

Shortened spine⁹⁴ Veratrum californicum: cattle; decreased coccygeal vertebrae, arthrogryposis Parbendazole toxicity: compression/fusion vertebrae, absence various limb bones, curvature long bones

Scoliosis, kyphosis, torticollis^81,83
–85 Wild lupins: see also craniofacial defects and shortened limbs Conium maculatum: cattle, sheep, pigs; plus arthrogryposis, carpal flexure, cleft palate Nicotiana tabacum, N. glauca: cattle/pigs; plus arthrogryposis, brachygnathia Locoweed (Astragalus, Oxytropis): cattle/sheep; also brachygnathia Lathyrus, Vicia: cattle/sheep; plus arthrogryposis, rotation forelimbs

Manganese deficiency³⁴ Cattle; congenital spinal stenosis and premature closure of growth plates associated with Mn deficiency but not proven

Limb abnormalities

Syndactyly^54,64 Autosomal recessive with incomplete penetrance and variable expression in Holstein-Friesian, Angus, Chianina, Hereford, Simmental, German Red Pied, Indian Hariana, and Japanese native cattle Right forelimb most commonly involved, but all limbs may be affected Veratrum californicum toxicity in cattle

Angular limb deformity^24,85,86 Trachymene spp: sheep; outward bowing particularly of forelimbs Locoweed (Astragalus, Oxytropis): cattle/sheep; also limb contractures, osteoporosis

Congenital hyperostosis^35,41 Severe edema: soft tissues of limbs (particularly antebrachium) Radiating trabeculae of new periosteal bone

Tibial hemimelia^62,79 Bilateral tibial hemimelia, cryptorchidism, ventral abdominal hernia, ± meningocele

Abnormalities in multiple axial and appendicular bones

Spider lamb syndrome^14,78,87,106 Excessively long limbs and neck leading to angular limb deformities, scoliosis/kyphosis, sternal deformities, Roman nose, degenerative joint disease Multiple irregular islands of ossification in epiphyseal cartilage

Schmallenberg virus, Akabane virus, ± other bunyaviruses^{1,39,47,48,108} Arthrogryposis (Schmallenberg, Akabane); torticollis, kyphosis, scoliosis, lordosis (Schmallenberg); brachygnathia inferior (Schmallenberg); and central nervous system abnormalities (Schmallenberg, Akabane, bluetongue virus)

Plant toxins ⁸³ Wild lupins, Conium maculatum, Nicotiana spp

Parbendazole toxicity See shortened spine

Methods of Mutation Detection

In cases where nutritional, toxic, and infectious causes of the skeletal abnormalities are considered unlikely or can be excluded, investigation of a genetic etiology should be considered. Historically, this would involve a breeding trial aimed at producing affected animals. This is most efficiently achieved by mating the sire of affected animals to dams that also sired affected animals. However, more long-term trials may be required whereby the sire is mated to unrelated dams and then mated to the F₁ females the following year. If the disease has recessive inheritance, it would be expected that 12.5% of the progeny in the second year would be affected.

Mutation detection, while easier than it was even 5 years ago, is still challenging, but potential exists for detection of a mutation without the expense, inherent variability, and potential welfare concerns involved in breeding defective animals. The type of gene mutation analysis used may vary depending on the inheritance pattern, determined from either pedigree analysis or breeding trials. With the decreasing costs of DNA technology, the temptation exists to neglect a thorough clinical and pathologic description of the disease. However, the recognition and characterization of new genetic diseases are often based on the reporting of specific abnormalities in a small number of animals on widely separated properties, sometimes over a period of several years, and it is important that veterinary clinicians and pathologists continue to accurately identify and record their findings.

Despite advances in technology, even in human medicine for nearly half of the described monogenic disorders, the molecular basis of the disease is still unknown.⁹⁶ There are a number of different methods of mutation detection, the 3 most commonly used methods are each discussed in turn.

Candidate Gene Approach

This technique is used when the clinical signs and histologic changes are consistent with a previously described disease of the same species with a known mutation or when the disease is closely analogous to a human disease or mouse model with a known affected gene.²⁸ The candidate gene can be sequenced either in one hit—using long-range polymerase chain reaction and next-generation sequencing—or by sequencing each exon of the gene individually. Although exonic sequencing may miss disease-causing intronic mutations, such as splice site mutations, this risk can be mitigated by including in the sequencing 100 to 200 base pairs of intron on either side of the exon. DNA of a poorer quality, such as that obtained from formalin-fixed paraffin-embedded sections, is suitable for this approach.

We used this approach to investigate a chondrodysplasia in Scottish Highland cattle. The mating of 2 purebred Scottish Highland cattle resulted in the birth of a calf with severely shortened limbs, a ventral abdominal hernia, domed head, shortened muzzle, and protruding tongue, consistent with bulldog chondrodysplasia of Dexter cattle. On histologic examination, intramembranous bone formation was normal, but endochondral ossification was severely restricted (Fig. 5). Testing showed that the calf was homozygous for the Dexter BD1 mutation,²¹ while both parents were heterozygous carriers.³⁰

Figure 5–6.
Bulldog chondrodysplasia, radius, bovine. Severely restricted endochondral ossification (arrows) with a disproportionate amount of bone formed by intramembranous ossification beneath the periosteum (arrowheads). HE. Figure 6. Inherited rickets, sheep; 2-year-old Corriedale sheep showing severe varus deformity of the forelimbs.

Single-Nucleotide Polymorphism Array and Genomewide Association Studies

Single-nucleotide polymorphisms (SNPs) are single base pair changes in DNA that occur commonly in the genome.¹⁹ The SNPs may either be silent (ie, cause no change in the amino acid sequence) or lead to functional changes if, for example, there is a change in an amino acid or if they affect a mRNA splice site. Genome-wide association studies make use of the concept of linkage disequilibrium, which, at its most basic, is where sections of DNA containing 2 or more SNPs are transferred together during a recombination event in meiosis and the sequence of DNA is inherited intact.^19,61 These areas of linkage disequilibrium become smaller with increasing numbers of generations. Using homozygosity mapping (in the case of recessive inheritance), we can determine regions that are inherited by descent (IBD) and associate them with the affected phenotype, thus focusing the area of DNA that must be searched for a mutation.

This technique requires a relatively large amount of high-quality DNA. While it is possible to do SNP arrays on formalin-fixed paraffin-embedded tissue without optimization, base calling may be decreased, thereby reducing the effectiveness of the tool. Chips are available for a number of different species. For cattle, Illumina arrays are available for a number of different levels of coverage across the bovine genome. Illumina beadchips are also available for the dog, sheep, and pig. Affymetrix, the other major SNP array producer, manufactures a SNP array for the cow, chicken, and dog.

We used this technique to determine the mutation causing inherited rickets in Corriedale sheep (Fig. 6). With use of the Illumina ovine 50-K chip and a combination of known heterozygotes, homozygous affected animals, and sire and dam DNA followed by homozygosity mapping, a large 6-Mb IBD region was found on OAR 6. By aligning this area with the annotated bovine genome, 35 potential candidate genes were found. Of the 35 genes in the area, several had an association with bone development or skeletal dysplasias; however, mutations in one of these—dentin matrix protein 1 (DMP1)—had recently been described as causing autosomal hypophosphatemic rickets in humans and mice.⁶⁸ Sequencing of this gene in our sheep demonstrated a nonsense mutation in exon 6 of the DMP1 gene, which led to a premature stop codon and truncation of the protein.¹¹⁴

The chances of successfully detecting a mutation using this technique is increased if the inheritance of the disease is known and if DNA from family members, including unaffected siblings, dams, and sires is available. Accurate recognition of phenotype is also important, as misdiagnosis of affected animals will obviously affect the analysis. Cost may be a limiting factor, as is the availability of tissue samples from related animals in diseases that take time to develop if sires, siblings, or dams are no longer available. The use of SNP chips for investigating diseases with dominant inheritance is more challenging. Not only are more affected animals required in the analysis, but in diseases with incomplete penetrance, accurate determination of affected animals can be difficult.

For cattle, a custom SNP chip that contains the genetic diseases of cattle with known mutations has been developed by Irish researchers. Known as the International Dairy and Beef chip, this provides the ability to scan bovine DNA samples for all known genetic diseases at one time.⁷²

Whole Exome Sequencing or Whole Genome Sequencing

Whole exome sequencing involves selectively sequencing only the coding sequences or exons in the genome by the process of exome capture (see Bamshad et al for a description of the process¹²), which allows enrichment of the sample. This may be a more cost-effective method for mutation detection than whole genome sequencing, particularly for Mendelian disorders. However, whole exome sequencing is not an option for all species. Exome capture is available for cattle, and an exome-wide DNA capture based on Bos taurus has been used for Bos indicus and Bison species,²⁶ indicating that there may be enough homology between some ruminant species and the bovine exome to allow its use in other ruminants.

A large quantity of good-quality DNA is required for exome sequencing, which may limit its usefulness in archived samples. In addition, approximately 5% to 10% of the exome (in humans) is poorly covered by the exome capture process.¹²

Whole genome sequencing, as the name suggests, involves sequencing the whole genome, including exons and introns, and the technology is now relatively affordable. Although it might seem that whole genome sequencing is the ultimate answer for determining a disease-causing mutation, there are limitations. In reality, sequencing of the “whole” genome is not necessarily possible, and the results may be skewed by poor coverage of GC-rich areas.¹¹ A large volume of information is obtained from whole genome sequencing, and appropriate computing and bioinformatics resources are required to process and interpret the data.

In a recent investigation of primordial dwarfism in Angus cattle, we have used SNP array with genome-wide association studies, followed by whole genome sequencing. Due to the distant familial relationships of affected animals, large numbers of small IBD regions were obtained on SNP array, and since many older samples were only formalin-fixed paraffin-embedded sections, base calling on these was unreliable. Basic analysis of whole genome sequencing from 2 affected animals produced a large number of variants between affected animals and the reference bovine genome, and data analysis is continuing.

One investigation in which this technique of combined SNP array and whole genome sequencing has been used successfully is an investigation into a chondrodysplasia in Grey Alpine cattle. SNP array and genome-wide association studies showed a 1.6-Mb IBD region on bovine chromosome 6, and whole genome sequencing of an affected calf revealed a 2–base pair deletion in the Ellis van Creveld 2 (EVC2) gene.⁷³

Conclusion

Distinguishing between genetic and nongenetic causes of congenital skeletal abnormalities in farmed livestock remains a challenge but has been greatly enhanced in recent years by advances in molecular genetic technology. Many techniques previously used only for research purposes because of their complexity and expense are now becoming available for commercial use. This trend will inevitably continue, but progress in molecular methods must be underpinned by accurate diagnosis and description of disease syndromes based on traditional methods, including epidemiology, clinical characteristics, pathology, and imaging. Pathologists will continue to play an important role in the recognition of these disorders and are ideally placed to recommend what techniques should be applied in an effort to make a definitive diagnosis.

Differential Diagnoses	Clinical Characteristics and Pathology
Osteogenesis imperfecta^6,10,51	May occur as “outbreaks” due to new dominant mutations in germline cells of sire; not breed associated Recessive inheritance in Barbados blackbelly sheep Often born dead or die within first few weeks of life Tissues affected: bones, tendons, eyes, teeth, ± skin Blue sclera, pink teeth, tendon and ligament hyperlaxity, intrauterine bone fractures, ± skin fragility in sheep Bones usually normal in shape Thin layer of abnormally basophilic osteoid lining cartilage spicules, lack of compaction of osteons in a cortex that consists of basophilic woven bone, trabecular microfractures
Osteopetrosis^{15,43,65,77,93}	Recessive inheritance Breeds: Angus (black and red), Simmental, Belgian Blue, and Hereford cattle; Polypay sheep May be born premature and smaller in size Often born dead or die within first few weeks of life Bones less fragile than in osteogenesis imperfecta May have a domed head, brachygnathia inferior, and impacted molars Cones of persistent primary spongiosa extending into the diaphysis Osteoclasts may be absent or abundant, depending on the underlying genetic defect
Intrauterine copper deficiency^42,97,102	May be associated with other manifestations of copper deficiency in older animals (eg, swayback) Liver copper concentrations not necessarily decreased at the time of examination; bone formed during a period of copper deficiency will remain fragile even after copper concentrations have returned to normal Bones in lambs, calves, and pigs may be osteopenic with narrow cortices and decreased deposition of osteoid on cartilage spicules, resembling the scorbutic lattice of vitamin C deficiency In calves and pigs, physeal thickening due to enlargement of the hypertrophic zone and the presence of trabecular microfractures in the primary spongiosa may resemble rickets
Inherited rickets^32,40,66	Autosomal recessive in Corriedale sheep; autosomal dominant or X-linked inheritance reported in humans Hypocalcemia and hypophosphatemia Depending on the type of genetic defect 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D may be decreased, normal or increased Thickened physes in long bones, enlarged costochondral junctions (rachitic rosary) Increased thickness of hypertrophic zone in physes of rapidly growing bones, trabecular microfractures, disorganization of the primary spongiosa, poorly mineralized seams of osteoid lining trabeculae and filling Haversian systems in cortices
Inherited scurvy⁵³	Autosomal recessive in Danish od/od pig Decreased plasma ascorbic acid concentrations Fragile bones, subperiosteal and joint hemorrhages, petechiae, and bruising of the skin Sparse osteoblasts, osteoid absent from cartilage spicules in the primary spongiosa (scorbutic lattice) Cortices thin, physes narrow
Arachnomelia^18,100	Simmental, German Fleckvieh, Italian, and Swiss brown cattle Brachygnathia inferior with a turned up maxilla Long bone cortices normal in thickness but diaphyses abnormally thin Severe hyperextension of fetlocks, with kyphosis and scoliosis

Differential Diagnoses	Clinical Characteristics and Pathology
Chondrodysplasia: genetic^{3,45,60,71,73}	Usually has recessive inheritance, so only small numbers of animals affected Breeds: Angus, Dexter, Japanese Brown, Tyrolean Grey, and Holstein/Friesian cattle; Texel, Merino, and Cabugi sheep; Danish Landrace pigs Clinical signs have a consistent presentation typically with minimal variation Normal liver zinc and manganese concentrations Shortened long bones (mild to severe depending on the gene affected), epiphyses potentially mushroomed, domed head, ± brachygnathia inferior Decreased thickness of physes, particularly hypertrophic zone; histologic lesions present in severe forms but mild or nonexistent in other forms
Chondrodysplasia: nutritional^34,44,49,107	Prevalence generally higher than expected for inherited forms of chondrodysplasia May be a history of drought or adverse weather events during pregnancy and exposure to pregnant animals to unusual supplementary feeding or toxic plants Variation in severity of dwarfism and twisted limbs; animals with mild cases improve after birth Shortened long bones, epiphyses may be mushroomed Decreased thickness of physes, particularly hypertrophic zone; histologically lesions may be mild or nonexistent Low liver zinc or manganese concentrations; if deficiency occurred for a finite period during gestation, zinc or manganese concentrations may have returned to normal
Plant toxins^{16,58,63,82,110}	Veratrum californicum toxicity in sheep: shortening metacarpal/metatarsal ± other bones, fusion metacarpal bones, arthrogryposis, hypermobility hock joints Wild lupins (Lupinus spp): cattle; shortening and rotation of limb bones, flexion contracture, arthrogryposis
Vitamin A toxicity^{20,33,69,89,112}	Pigs: shortening of long bones, abnormally shaped long bones and carpal/tarsal bones, osteoporosis Calves: possibly associated with “hyena disease”; premature closure of hind limb growth plates, segmental narrowing of physes

Differential Diagnoses	Clinical Characteristics and Pathology
Abnormal head shape
Chondrodysplasia	See Table 3
Osteopetrosis⁸⁰	See Table 2; markedly thickened skull bones that in some animals may contain cystic cavities, impacted molars
Craniosynostoses²⁷	Possibly autosomal dominant with incomplete penetrance in Cabugi sheep; domed head, protruding mandible, exophthalmos, dwarfism
Toxic plants^{57,85,88,91,110}	Veratrum californicum: sheep, cattle; cyclopia, harelip/cleft palate, mandibular micrognathia, tracheal/laryngeal stenosis Wild lupins (Lupinus spp): cattle; cleft palate, brachygnathia superior Prunus serotina (wild black cherry): pigs; cleft palate, arthrogryposis Datura stramonium: pigs; cleft palate, arthrogryposis, although definitive evidence lacking Leucaena, Mimosa: cattle/sheep; cleft palate, brachygnathia, agnathia, harelip, cranioschisis, forelimb flexure, scoliosis/kyphosis/torticollis
Vitamin A deficiency¹⁰¹	Cattle/pigs; failure of cranial cavity nerve foramina and spinal canal to enlarge
Vertebral abnormalities
Brachyspina^7,8,99	Cattle; smaller than normal size, shortened vertebral column due to fusion of vertebrae, and long thin limbs Irregular areas of ossification separated by cartilage, fusion of epiphyses, and diaphysis of adjacent vertebrae
Complex vertebral malformation^2,4,5	Cattle; smaller than normal size, consistent arthrogryposis of forelimbs ± hind limbs, shortening of cervical and thoracic vertebral column, hemivertebrae, fused vertebrae, scoliosis Heart defects in approximately 50% of affected animals
Shortened spine⁹⁴	Veratrum californicum: cattle; decreased coccygeal vertebrae, arthrogryposis Parbendazole toxicity: compression/fusion vertebrae, absence various limb bones, curvature long bones
Scoliosis, kyphosis, torticollis^81,83 –85	Wild lupins: see also craniofacial defects and shortened limbs Conium maculatum: cattle, sheep, pigs; plus arthrogryposis, carpal flexure, cleft palate Nicotiana tabacum, N. glauca: cattle/pigs; plus arthrogryposis, brachygnathia Locoweed (Astragalus, Oxytropis): cattle/sheep; also brachygnathia Lathyrus, Vicia: cattle/sheep; plus arthrogryposis, rotation forelimbs
Manganese deficiency³⁴	Cattle; congenital spinal stenosis and premature closure of growth plates associated with Mn deficiency but not proven
Limb abnormalities
Syndactyly^54,64	Autosomal recessive with incomplete penetrance and variable expression in Holstein-Friesian, Angus, Chianina, Hereford, Simmental, German Red Pied, Indian Hariana, and Japanese native cattle Right forelimb most commonly involved, but all limbs may be affected Veratrum californicum toxicity in cattle
Angular limb deformity^24,85,86	Trachymene spp: sheep; outward bowing particularly of forelimbs Locoweed (Astragalus, Oxytropis): cattle/sheep; also limb contractures, osteoporosis
Congenital hyperostosis^35,41	Severe edema: soft tissues of limbs (particularly antebrachium) Radiating trabeculae of new periosteal bone
Tibial hemimelia^62,79	Bilateral tibial hemimelia, cryptorchidism, ventral abdominal hernia, ± meningocele
Abnormalities in multiple axial and appendicular bones
Spider lamb syndrome^14,78,87,106	Excessively long limbs and neck leading to angular limb deformities, scoliosis/kyphosis, sternal deformities, Roman nose, degenerative joint disease Multiple irregular islands of ossification in epiphyseal cartilage
Schmallenberg virus, Akabane virus, ± other bunyaviruses^{1,39,47,48,108}	Arthrogryposis (Schmallenberg, Akabane); torticollis, kyphosis, scoliosis, lordosis (Schmallenberg); brachygnathia inferior (Schmallenberg); and central nervous system abnormalities (Schmallenberg, Akabane, bluetongue virus)
Plant toxins ⁸³	Wild lupins, Conium maculatum, Nicotiana spp
Parbendazole toxicity	See shortened spine

Footnotes

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

References

Afonso

Abrahantes

Conraths

. The Schmallenberg virus epidemic in Europe: 2011–2013. Prev Vet Med. 2014;116:391–403.

Agerholm

Andersen

Almskou

. Evaluation of the inheritance of the complex vertebral malformation syndrome by breeding studies. Acta Vet Scand. 2004;45:133–137.

Agerholm

Arnbjerg

Andersen

. Familial chondrodysplasia in Holstein calves. J Vet Diagn Invest. 2004;16:293–298.

Agerholm

Bendixen

Andersen

. Complex vertebral malformation in holstein calves. J Vet Diagn Invest. 2001;13:283–289.

Agerholm

Bendixen

Arnbjerg

. Morphological variation of “complex vertebral malformation” in Holstein calves. J Vet Diagn Invest. 2004;16:548–553.

Agerholm

Lund

Bloch

. Osteogenesis imperfecta in Holstein-Friesian calves. Zentralbl Veterinarmed A. 1994;41:128–138.

Agerholm

McEvoy

Arnbjerg

. Brachyspina syndrome in a Holstein calf. J Vet Diagn Invest. 2006;18:418–422.

Agerholm

Peperkamp

. Familial occurrence of Danish and Dutch cases of the bovine brachyspina syndrome. BMC Vet Res. 2007;3:8.

Arteaga-Solis

Sui-Arteaga

Kim

. Material and mechanical properties of bones deficient for fibrillin-1 or fibrillin-2 microfibrils. Matrix Biol. 2011;30:188–194.

10.

Arthur

Thompson

Swarbrick

. Lethal osteogenesis imperfecta and skin fragility in newborn New Zealand Romney lambs. N Z Vet J. 1992;40:112–116.

11.

Bai

Sartor

Cavalcoli

. Current status and future perspectives for sequencing livestock genomes. J Anim Sci Biotechnol. 2012;3:8.

12.

Bamshad

Bigham

. Exome sequencing as a tool for Mendelian disease gene discovery. Nat Rev Genet. 2011;12:745–755.

13.

Beever

Marron

, inventors; Agrigenomics Inc, assignee. Screening for the genetic defect causing tibial hemimelia in bovines. US patent 20090239212. April 17, 2012.

14.

Beever

Smit

Meyers

. A single-base change in the tyrosine kinase II domain of ovine FGFR3 causes hereditary chondrodysplasia in sheep. Anim Genet. 2006;37:66–71.

15.

Bildfell

Piripi

Thompson

. Osteopetrosis in Polypay lambs [abstract]. Paper presented at: 42nd Annual Western Conference of Veterinary Diagnostic Pathologists; 2009; Minneapolis, MN.

16.

Binns

Shupe

Keeler

. Chronologic evaluation of teratogenicity in sheep fed Veratrum californicum. J Am Vet Med Assoc. 1965;147:839–842.

17.

Buitkamp

Joerdis

Goetz

K-U

. Arachnomelia syndrome in Simmental cattle is caused by a homozygous 2-bp deletion in the molybdenum cofactor synthesis step 1 gene (MOCS1). BMC Genet. 2011;12:11.

18.

Buitkamp

Luntz

Emmerling

. Syndrome of arachnomelia in Simmental cattle. BMC Vet Res. 2008;4:39.

19.

Bush

Moore

. Chapter 11: genome-wide association studies. PLoS Comput Biol. 2012;8:e1002822.

20.

Carroll Woodard

Donovan

Eckhoff

. Vitamin (A and D)-induced premature physeal closure (hyena disease) in calves. J Comp Pathol. 1997;116:353–366.

21.

Cavanagh

Tammen

Windsor

. Bulldog dwarfism in Dexter cattle is caused by mutations in ACAN. Mamm Genome. 2007;18:808–814.

22.

Charlier

Agerholm

Coppieters

. A deletion in the bovine FANCI gene compromises fertility by causing fetal death and brachyspina. PLoS One. 2012;7:e43085.

23.

Chavez

Serda

Choe

. Molecular basis for pseudo vitamin D-deficiency rickets in the Hannover pig. J Nutr Biochem. 2003;14:378–385.

24.

Clark

Carlisle

Beasley

. Observations on the pathology of bent leg of lambs in South-Western Queensland. Aust Vet J. 1975;51:4–10.

25.

Cortes

Baria

Schwartz

. Sulfation of chondroitin sulfate proteoglycans is necessary for proper Indian hedgehog signaling in the developing growth plate. Development. 2009;136:1697–1706.

26.

Cosart

Beja-Pereira

Chen

. Exome-wide DNA capture and next generation sequencing in domestic and wild species. BMC Genomics. 2011;12:347.

27.

Dantas

Medeiros

Figueiredo

. Skeletal dysplasia with craniofacial deformity and disproportionate dwarfism in hair sheep of northeastern Brazil. J Comp Pathol. 2014;150:245–252.

28.

Dattani

. The candidate gene approach to the diagnosis of monogenic disorders. Horm Res. 2009;71(suppl 2):14–21.

29.

Dennis

. Perinatal lamb mortality in Western Australia. 7. Congenital defects. Aust Vet J. 1975;51:80–82.

30.

Dittmer

Hogan

Thompson

. A case of bulldog-type chondrodysplasia in a miniature Scottish highland calf. J Comp Pathol. 2013;148:98.

31.

Dittmer

Thompson

. Vitamin D metabolism and rickets in domestic animals: a review. Vet Pathol. 2011;48:389–407.

32.

Dittmer

Thompson

Blair

. Pathology of inherited rickets in Corriedale sheep. J Comp Pathol. 2009;141:147–155.

33.

Dobson

. Osteodystrophy associated with hypervitaminosis A in growing pigs. Aust Vet J. 1969;45:570–573.

34.

Doige

Townsend

Janzen

. Congenital spinal stenosis in beef calves in western Canada. Vet Pathol. 1990;27:16–25.

35.

Doize

Martineau

. Congenital hyperostosis in piglets: a consequence of a disorganization of the perichondrial ossification groove of Ranvier. Can J Comp Med. 1984;48:414–419.

36.

Drögemüller

Leeb

Harlizius

. Congenital syndactyly in cattle: four novel mutations in the low density lipoprotein receptor-related protein 4 gene (LRP4). BMC Genet. 2007;8:5.

37.

Drögemüller

Tetens

Sigurdsson

. Identification of the bovine arachnomelia mutation by massively parallel sequencing implicates sulfite oxidase (SUOX) in bone development. PLoS Genet. 2010;6:e1001079.

38.

Duchesne

Gautier

Chadi

. Identification of a doublet missense substitution in the bovine LRP4 gene as a candidate causal mutation for syndactyly in Holstein cattle. Genomics. 2006;88:610–621.

39.

Elliott

Brennan

. Emerging phleboviruses. Curr Opin Virol. 2014;5:50–57.

40.

Feldman

Malloy

. Mutations in the vitamin D receptor and hereditary vitamin D-resistant rickets. Bonekey Rep. 2014;3:510.

41.

Gibson

Rogers

. Congenital porcine hyperostosis. Aust Vet J. 1980;56:254–255.

42.

Gooneratne

Buckley

Christensen

. Review of copper deficiency and metabolism in ruminants. Can J Anim Sci. 1989;69:819–845.

43.

Greene

Leipold

Hibbs

. Congenital osteopetrosis in Angus calves. J Am Vet Med Assoc. 1974;164:389–395.

44.

Hansen

Spears

Lloyd

. Feeding a low manganese diet to heifers during gestation impairs fetal growth and development. J Dairy Sci. 2006;89:4305–4311.

45.

Harper

Latter

Nicholas

. Chondrodysplasia in Australian Dexter cattle. Aust Vet J. 1998;76:199–202.

46.

Hasan

Vogeli

Stoll

. Intragenic deletion in the gene encoding L-gulonolactone oxidase causes vitamin C deficiency in pigs. Mamm Genome. 2004;15:323–333.

47.

Hashiguchi

Nanba

Kumagai

. Congenital abnormalities in newborn lambs following Akabane virus infection in pregnant ewes. Natl Inst Anim Health Q (Tokyo). 1979;19:1–11.

48.

Herder

Wohlsein

Peters

. Salient lesions in domestic ruminants infected with the emerging so-called Schmallenberg virus in Germany. Vet Pathol. 2012;49:588–591.

49.

Hidiroglou

Ivan

Bryan

. Assessment of the role of manganese in congenital joint laxity and dwarfism in calves. Annal Rech Vet. 1990;21:281–284.

50.

Hirano

Matsuhashi

Kobayashi

. Identification of an FBN1 mutation in bovine Marfan syndrome-like disease. Anim Genet. 2012;43:11–17.

51.

Holmes

Baker

Davies

. Osteogenesis imperfecta in lambs. Vet Rec. 1964;76:980–984.

52.

Hudson

Taniguchi-Sidle

Boras

. Alx-4, a transcriptional activator whose expression is restricted to sites of epithelial-mesenchymal interactions. Dev Dyn. 1998;213:159–169.

53.

Jensen

Basse

Nielsen

. Congenital ascorbic acid deficiency in pigs. Acta Vet Scand. 1983;24:392–402.

54.

Johnson

Steffen

Lynch

. Defective splicing of Megf7/Lrp4, a regulator of distal limb development, in autosomal recessive mulefoot disease. Genomics. 2006;88:600–609.

55.

Jolly

Windsor

. Genetic diseases of cattle. In: Parkinson

Vermunt

Malmo

, eds. Diseases of Cattle in Australasia. Wellington, New Zealand: VetLearn; 2010:759–777.

56.

Jones

Jolly

. Dwarfism in Hereford cattle: a genetic morphological and biochemical study. N Z Vet J. 1982;30:185–189.

57.

Keeler

. Teratogens in plants. J Anim Sci. 1984;58:1029–1039.

58.

Keeler

Stuart

. The nature of congenital limb defects induced in lambs by maternal ingestion of Veratrum californicum. J Toxicol Clin Toxicol. 1987;25:273–286.

59.

Kirkland

Barry

Harper

. The development of Akabane virus-induced congenital abnormalities in cattle. Vet Rec. 1988;122:582–586.

60.

Koltes

Mishra

Kumar

. A nonsense mutation in cGMP-dependent type II protein kinase (PRKG2) causes dwarfism in American Angus cattle. Proc Natl Acad Sci U S A. 2009;106:19250–19255.

61.

LaFramboise

. Single nucleotide polymorphism arrays: a decade of biological, computational and technological advances. Nucleic Acids Res. 2009;37:4181–4193.

62.

Lapointe

J-M

Lachance

Steffen

. Tibial hemimelia, meningocele, and abdominal hernia in shorthorn cattle. Vet Pathol. 2000;37:508–511.

63.

Lee

Cook

Panter

. Lupine induced “crooked calf disease” in Washington and Oregon: identification of the alkaloid profiles in Lupinus sulfureus , Lupinus leucophyllus, and Lupinus sericeus. J Agric Food Chem. 2007;55:10649–10655.

64.

Leipold

Adrian

Huston

. Anatomy of hereditary bovine syndactylism: I. Osteology. J Dairy Sci. 1969;52:1422–1431.

65.

Leipold

Doige

Kaye

. Congenital osteopetrosis in Aberdeen Angus calves. Can Vet J. 1970;11:181–185.

66.

Levine

Kleeman

Felsenfeld

. The journey from vitamin D–resistant rickets to the regulation of renal phosphate transport. Clin J Am Soc Nephrol. 2009;4:1866–1877.

67.

Pawlik

Elcioglu

. LRP4 mutations alter Wnt/beta-catenin signaling and cause limb and kidney malformations in Cenani-Lenz syndrome. Am J Hum Genet. 2010;86:696–706.

68.

Lorenz-Depiereux

Bastepe

Benet-Pages

. DMP1 mutations in autosomal recessive hypophosphatemia implicate a bone matrix protein in the regulation of phosphate homeostasis. Nat Genet. 2006;38:1248–1250.

69.

MacKay

Woodard

Donovan

. Focal premature physeal closure (hyena disease) in calves. J Am Vet Med Assoc. 1992;201:902–905.

70.

Meyer

McDaneld

Swist

. A deletion mutation in bovine SLC4A2 is associated with osteopetrosis in Red Angus cattle. BMC Genomics. 2010;11:337.

71.

Moritomo

Ishibashi

Miyamoto

. Morphological changes of epiphyseal plate in the long bone of chondrodysplastic dwarfism in Japanese brown cattle. J Vet Med Sci. 1992;54:453–459.

72.

Mullen

McClure

Kearney

. Development of a custom SNP chip for dairy and beef cattle breeding, parentage and research. Paper presented at: Proceedings of the Interbull Meeting; August 23–25, 2013; Nantes, France.

73.

Murgiano

Jagannathan

Benazzi

. Deletion in the EVC2 gene causes chondrodysplastic dwarfism in Tyrolean grey cattle. PLoS One. 2014;9:e94861.

74.

Nicholas

. Online Mendelian Inheritance of Animals, University of Sydney. http://omia.angis.org.au/home/. Accessed November 13, 2014.

75.

Nielsen

Bendixen

Arnbjerg

. Abnormal growth plate function in pigs carrying a dominant mutation in type X collagen. Mamm Genome. 2000;11:1087–1092.

76.

Nistala

Lee-Arteaga

Smaldone

. Fibrillin-1 and -2 differentially modulate endogenous TGF-beta and BMP bioavailability during bone formation. J Cell Biol. 2010;190:1107–1121.

77.

O’Toole

Swist

Steadman

. Neuropathology and craniofacial lesions of osteopetrotic red angus calves. Vet Pathol. 2012;49:746–754.

78.

Oberbauer

East

Pool

. Developmental progression of the spider lamb syndrome. Small Ruminant Res. 1995;18:179–184.

79.

Ojo

Guffy

Saperstein

. Tibial hemimelia in Galloway calves. J Am Vet Med Assoc. 1974;165:548–550.

80.

Ojo

Leipold

Cho

. Osteopetrosis in two Hereford calves. J Am Vet Med Assoc. 1975;166:781–783.

81.

Panter

Bunch

Keeler

. Multiple congenital contractures (MCC) and cleft palate induced in goats by ingestion of piperidine alkaloid-containing plants: reduction in fetal movement as the probable cause. J Toxicol Clin Toxicol. 1990;28:69–83.

82.

Panter

Gardner

Molyneux

. Teratogenic and fetotoxic effects of two piperidine alkaloid-containing lupines (L. formosus and L. arbustus) in cows. J Nat Toxins. 1998;7:131–140.

83.

Panter

James

Gardner

. Lupines, poison-hemlock and Nicotiana spp: toxicity and teratogenicity in livestock. J Nat Toxins. 1999;8:117–134.

84.

Panter

Keeler

Bunch

. Congenital skeletal malformations and cleft palate induced in goats by ingestion of Lupinus , Conium and Nicotiana species. Toxicon. 1990;28:1377–1385.

85.

Panter

Welch

Gardner

. Poisonous plants: effects on embryo and fetal development. Birth Defects Res C Embryo Today. 2013;99:223–234.

86.

Philbey

. Trachymene glaucifolia associated with bentleg in lambs. Aust Vet J. 1990;67:468.

87.

Phillips

Bunn

Anderson

. Ovine hereditary chondrodysplasia (spider syndrome) in Suffolk lambs. Aust Vet J. 1993;70:73–74.

88.

Pimentel

Correa

Gardner

. Mimosa tenuiflora as a cause of malformations in ruminants in the northeastern Brazilian semiarid rangelands. Vet Pathol. 2007;44:928–931.

89.

Pryor

Seawright

McCosker

. Hypervitaminosis A in the pig. Aust Vet J. 1969;45:563–569.

90.

Qin

D’Souza

Feng

. Dentin matrix protein 1 (DMP1): new and important roles for biomineralization and phosphate homeostasis. J Dent Res. 2007;86:1134–1141.

91.

Riet-Correa

Medeiros

Schild

. A review of poisonous plants that cause reproductive failure and malformations in the ruminants of Brazil. J Appl Toxicol. 2012;32:245–254.

92.

Rousselle

Heymann

. Osteoclastic acidification pathways during bone resorption. Bone. 2002;30:533–540.

93.

Sartelet

Stauber

Coppieters

. A missense mutation accelerating the gating of the lysosomal Cl-/H+-exchanger ClC-7/Ostm1 causes osteopetrosis with gingival hamartomas in cattle. Dis Model Mech. 2014;7:119–128.

94.

Saunders

Shone

Philip

. The effects of methyl-5(6)-butyl-2-benzimidazole carbamate (parbendazole) on reproduction in sheep and other animals: I. Malformations in newborn lambs. Cornell Vet. 1974;64(suppl 4):7–40.

95.

Smith

Dally

Sainz

. Enhanced skeletal growth of sheep heterozygous for an inactivated fibroblast growth factor receptor 3. J Anim Sci. 2006;84:2942–2949.

96.

Statistics OMIM. http://www.omim.org/statistics/entry, Accessed July 15, 2014.

97.

Suttle

Angus

Nisbet

. Osteoporosis in copper-depleted lambs. J Comp Pathol. 1972;82:93–97.

98.

Takeda

Takami

Oguni

. Positional cloning of the gene LIMBIN responsible for bovine chondrodysplastic dwarfism. Proc Natl Acad Sci U S A. 2002;99:10549–10554.

99.

Testoni

Diana

Olzi

. Brachyspina syndrome in two Holstein calves. Vet J. 2008;177:144–146.

100.

Testoni

Gentile

. Arachnomelia in four Italian brown calves. Vet Rec. 2004;155:372.

101.

Thompson

. Bones and joints. In: Maxie

, ed. Jubb, Kennedy, and Palmer’s Pathology of Domestic Animals. 5th ed. Philadelphia, PA: Elsevier Saunders; 2007:1–184.

102.

Thompson

Audige

Arthur

. Osteochondrosis associated with copper deficiency in young farmed red deer and wapiti x red deer hybrids. N Z Vet J. 1994;42:137–143.

103.

Thompson

Blair

Linney

. Chondrodysplasia of Texel sheep: a new disease of suspected genetic aetiology. N Z Vet J. 2003;51:45–46.

104.

Thomsen

Horn

Panitz

. A missense mutation in the bovine SLC35A3 gene, encoding a UDP-N-acetylglucosamine transporter, causes complex vertebral malformation. Genome Res. 2006;16:97–105.

105.

Titus

Yan

Wilson

. The Fanconi anemia/BRCA gene network in zebrafish: embryonic expression and comparative genomics. Mutat Res. 2009;668:117–132.

106.

Troyer

Thomas

Stein

. A morphologic and biochemical evaluation of the spider syndrome in Suffolk sheep. Anat Histol Embryol. 1988;17:289–300.

107.

Valero

Alley

Badcoe

. Chondrodystrophy in calves associated with manganese deficiency. N Z Vet J. 1990;38:161–167.

108.

van den Brom

Luttikholt

Lievaart-Peterson

. Epizootic of ovine congenital malformations associated with Schmallenberg virus infection. Tijdschr Diergeneeskd. 2012;137:106–111.

109.

Warman

Cormier-Daire

Hall

. Nosology and classification of genetic skeletal disorders: 2010 revision. Am J Med Genet A. 2011;155:943–968.

110.

Welch

Panter

Lee

. Cyclopamine-induced synophthalmia in sheep: defining a critical window and toxicokinetic evaluation. J Appl Toxicol. 2009;29:414–421.

111.

White

Windsor

. Congenital chondrodystrophy of unknown origin in beef herds. Vet J. 2012;193:336–343.

112.

Woodard

Donovan

Fisher

. Pathogenesis of vitamin (A and D)–induced premature growth-plate closure in calves. Bone. 1997;21:171–182.

113.

Wuyts

Cleiren

Homfray

. The ALX4 homeobox gene is mutated in patients with ossification defects of the skull (foramina parietalia permagna, OMIM 168500). J Med Genet. 2000;37:916–920.

114.

Zhao

Dittmer

Blair

. A novel nonsense mutation in the DMP1 gene identified by a genome-wide association study is responsible for inherited rickets in Corriedale sheep. PLoS ONE. 2011;6(7):e21739.

115.

Zhao

Onteru

Piripi

. In a shake of a lamb’s tail: using genomics to unravel a cause of chondrodysplasia in Texel sheep. Anim Genet. 2012;43:9–18.

Approach to Investigating Congenital Skeletal Abnormalities in Livestock

Abstract

Keywords

Clinical History

Examination of Affected Animals

Methods of Mutation Detection

Candidate Gene Approach

Single-Nucleotide Polymorphism Array and Genomewide Association Studies

Whole Exome Sequencing or Whole Genome Sequencing

Conclusion

Footnotes

Declaration of Conflicting Interests

Funding

References