Genetic Disorders

ANGELA M. HUGHES and KARI J. EKENSTEDT, Consulting Editors

nherited diseases and congenital defects are structural or functional abnormalities that may or may not be obvious at birth.

It is important to remember that not all congenital defects are genetic in origin, and making the distinction between conditions caused by an environmental factor or by a genetic cause will help clients make appropriate changes to prevent the disorder in the future. To determine the cause of a disorder, it is vital that each case be examined thoroughly. Each case should be subjected to a full, careful description of the disorder including a complete necropsy. The environment and management conditions should be investigated for potential causative agents, and all available genetic information for the affected animal(s) and unaffected herdmates should be collected including sex, birth date, and breed. Environmental teratogens affecting large animals include toxic plants, drugs, viruses, and physical agents (e.g., hyperthermia, irradiation). Although it may be difficult to identify inciting teratogens, analysis of affected herds will reveal patterns that follow seasonal or management changes or stressful events.

This chapter focuses on the essential genetic information that veterinarians require to inform their clients about genetic diseases, make breeding recommendations, and find information on diseases and traits with a genetic component.

Genetic Information

The basic blueprint for life in most organisms, the genetic material, is deoxyribonucleic acid (DNA). DNA is composed of two strands of nucleotides: adenine (A), thymine (T), guanine (G), and cytosine (C). These nucleotides, or bases, align to form complementary pairings such that A always pairs with T and G always pairs with C. These strands can be completely separated and replicated in preparation for cellular division. Alternatively, they can be temporarily partially separated and transcribed into ribonucleic acid (RNA), which is then translated into functional proteins.

The DNA sequence of a mammalian genome is divided into autosomes and a pair of sex chromosomes, X and Y. Each chromosome contains a variety of genes and “filler” DNA that does not code for proteins but may determine where and to what degree each gene is expressed. Genes on each chromosome code for the various structural and enzymatic proteins required for life. One set of chromosomes, and thus one set of genes, is inherited from each parent. As a result, a mammal generally has two copies, or alleles, of any given gene. Simple traits or diseases involving these genes can be inherited in dominant or recessive patterns and involve the autosomes or sex chro­mosomes. Not all traits or inherited diseases are simple; many involve multiple genes and interactions between genes and the environment, which can unfortunately make it difficult to diagnose and understand a complex disease or trait.

Recessive Inheritance

Traits or diseases with a recessive mode of inheritance require two affected alleles of the gene to express that trait or disease. These conditions can be autosomal, involving genes on the autosomes, or sex-linked, caused by genes on either the X or Y chromosome. Generally, autosomal recessive traits or diseases are caused by loss-of-function mutations in which the gene product has decreased ability or no ability to perform its original function. When an animal inherits the affected allele from both parents, it is homozygous for the affected alleles and is unable to make functionally normal copies of that specific protein.

The hallmarks of autosomal recessive traits include 1) all offspring of two affected parents are affected, 2) approximately equal numbers of males and females are affected, and 3) unaf­fected carrier parents can produce affected offspring, and thus the trait or disease can skip generations. In addition, mating an affected individual to a noncarrier (homozygous normal) produces only offspring that appear normal but carry the affected allele. Breeding two carrier individuals results in approximately 25% normal, 50% carrier, and 25% affected offspring.

An example of an autosomal recessive trait is spider lamb syndrome, or hereditary chondrodysplasia. Lambs affected with this syndrome display several skeletal abnormalities including disproportionately long “spider” legs, curvature of the spine, facial deformities, rib and sternum deformities, lack of body fat, and muscular atrophy. The causative mutation is a single base-pair substitution, from a T to an A, altering a highly conserved and nonpolar valine amino acid to a polar glutamate in the critical tyrosine kinase II region of the fibroblast growth factor receptor 3 (FGFR-3) gene. This prevents the receptor from functioning to limit endochondral ossification and thus bone growth.¹ Genetic testing for the causative mutation has helped limit the prevalence of affected lambs; however, they are still occasionally observed, particularly where genetic testing has not been routinely used.²

Dominant Inheritance

Traits or diseases with a dominant mode of inheritance only require one affected allele to express the trait or disease, also known as the affected phenotype, and there is no distinguishable difference in the affected phenotype of a heterozygote and homozygote (homozygous for the affected allele).

The main feature of an autosomal dominant trait or disorder is that affected individuals must have at least one affected parent (unless it is a new mutation), and thus the disorder does not skip generations. There is no sex bias with an autosomal dominant trait, and approximately half the offspring of a heterozygous affected individual bred to a normal individual will be affected. Breeding two heterozygous affected individuals will result in 75% affected (one third of which are homozygous and two thirds of which are heterozygous) and 25% normal offspring.

An example of an autosomal dominant disease is myotonia in goats. When startled or when making sudden, forceful move­ments, these goats can develop severe, acute muscle stiffness causing immobility and sometimes falling over, resulting in descriptions such as “fainting,” “nervous,” “stiff-legged,” or “epileptic” goats. A single nucleotide change, from a G to a C, was identified that substituted a proline amino acid for a conserved alanine in the CLCN1 gene, a chloride channel in the muscle fibers. This alteration in the chloride channel causes a diminished channel-open probability at voltages near the resting membrane potential of skeletal muscle, resulting in decreased chloride conductance and a significantly decreased electrical threshold for firing action potentials. Ultimately, this altered chloride channel allows conduction of repetitive impulses that result in sustained muscle fiber contraction and stiffness.³ Far from selecting against this trait, these myotonic or “fainting” goats are purposely bred in some scenarios, where they are generally used for meat production; in fact, they are considered by some to be a breed in their own right.

Codominant Inheritance

The codominant (or semidominant) pattern of inheritance is distinguished by the fact that homozygotes can be differentiated from heterozygotes on the basis of clinical features. A well- known example of a codominant disease is hyperkalemic periodic paralysis (HYPP) in Quarter Horses. In HYPP the voltage-gated sodium channels in the muscle fibers have a mutation, specifi­cally a C to G change that substitutes a leucine residue for a highly conserved phenylalanine, which increases sodium perme­ability across the skeletal muscle cell membrane. This causes increased muscle mass but also drooling, prolapse of the nictitating membrane (“third eyelid”), respiratory stridor, and weakness. Homozygous HYPP horses experience more frequent and severe clinical signs of disease than heterozygous horses.^4,5 Thus it is important to counsel breeders to avoid producing homozygous affected horses by not mating two heterozygous horses, since approximately 25% of their offspring would be expected to be homozygous affected and 50% would be het­erozygous, while only 25% would be homozygous normal. Breeding a heterozygous horse to an unaffected horse will also result in approximately 50% of the offspring being heterozygous with no possibility of producing a homozygous affected foal. It should be pointed out that HYPP heterozygous horses are still considered affected; however, their disease is easier to manage environmentally (via nutrition and management practices) compared with HYPP homozygous-affected horses.

Sex-Linked Traits

Sex-linked traits and diseases involve genes located on either the X or Y chromosome. Males have only one copy each of the X and Y chromosomes, so they are hemizygous. As a result, males are particularly susceptible to sex-linked conditions because if they have a mutant allele for one of the genes on either of the sex chromosomes, they lack a “backup” copy of the normal allele and will express the affected phenotype. Females generally are less likely to demonstrate sex-linked recessive traits or diseases because they have two copies of the X chromosome.

Key characteristics of X-linked recessive traits are 1) the trait appears with much greater frequency in males than females, 2) half the sons of carrier females will be affected, 3) half the daughters of carrier females will be carriers, and 4) all of the daughters of affected males will be carriers. Hemophilia A, characterized by a strong tendency to bleed resulting from mutations in the clotting factor VIII gene on the X chromosome, is an example of an X-linked recessive disorder.

X-linked dominant traits are characterized by the following: 1) affected offspring must have at least one affected parent, 2) the disorder does not skip generations, and 3) an affected male mated to normal females will transmit the mutation to all his daughters but not to his sons.

Few Y-linked diseases are recognized in any mammalian species because of the small number of genes on the Y chromo­some. In addition, the majority of the genes contained on the Y chromosome are involved in male fertility; thus mutations in these genes generally render the animal sterile, resulting in no transmission of the mutations to future generations.

Polygenic Traits

In addition to the single-gene traits and diseases, numerous conditions are the result of two or more genes and may also involve genetic and environmental interactions. In fact, entire biological pathways can be involved and interconnected, influenced by gene-gene interactions and/or gene-environment interactions; such pathways can turn genes, or groups of genes, on or off. Many of these polygenic traits are economically important. For example, a large number of genes may be contributing a small portion to the overall milk production of a dairy cow,⁶ and endocrine fertility traits in dairy cattle are likewise driven by multiple genetic loci.⁷ In beef cattle, at least eight genes have been significantly associated with stature and thus the physiology of growth.⁸ Other highly polygenic beef cattle traits include carcass and meat quality.⁹

Now that the major large animal species have had their genomes sequenced, cattle and sheep livestock associations and producers are increasingly using whole-genome approaches for genetic evaluation, in an effort to tackle polygenic traits, as well as overall genetic value. For example, dairy cattle geneticists are now using single nucleotide polymorphism (SNP) markers spanning the entire genome, combined with progeny test data, in order to predict genetic merit for polygenic traits for any animal. This can be done before the animal produces any offspring. The swine industry is also using genome-wide data; however, much of these data are proprietary to specific corporations, so they may be difficult to access.

Fueled by decreasing costs, whole-genome sequencing is now becoming broadly available; this allows identification of additional forms of genetic variation such as copy number variations and short insertions and deletions. With data now consisting of entire genomes, more accurate breeding values can be attained, a better biological understanding of the genome can be achieved,¹⁰ and areas of the genome that impact production—even if the physiology is unknown—can be taken into consideration.

Teasing out the details of a complex polygenic system, such as assigning a specific gene and a measure of its contribution to a polygenic trait like milk production or meat quality, is difficult; however, research is ongoing to identify the exact genes involved and the role each gene plays for many economi­cally important traits and diseases. Thus it is anticipated that our understanding of polygenic traits in large animals will continue to increase in the next few years. Companies are already beginning to explore the value of genetic predictions for disease susceptibility and offer products that assist in genetic selection of, for example, dairy heifers, based on various health and disease traits. The full utility of such products is still being evaluated.

Penetrance and Expressivity

For many simple traits, there is an obvious difference between the two phenotypes that can be translated to the alleles involved, or genotype, with 100% certainty. A good example of this is albinism, which is considered to be 100% penetrant, with penetrance defined as the percentage of individuals within a given genotype that actually show the phenotype associated with that genotype. Influences such as modifying genes or environmental interactions can alter the expression of certain genes such that the exact genotype is not expressed in the outward phenotype. Alternatively, the function of a gene may be subtle, making it difficult to measure distinctions adequately between genotypes. Genotypes that may not be expressed in every individual are considered to be “incompletely penetrant.”

Another measure of genetic expression is the concept of expressivity. Modifier genes and environmental influences can affect the degree to which a genotype is expressed. For example, “variable expressivity” may be involved in pigment intensity when two individuals have the same genotype at the gene responsible for red pigmentation, but one has clearly darker red hairs than the other.

Incomplete penetrance and variable expressivity can greatly complicate analysis of genetic traits and diseases. These factors can also make breeding decisions more difficult because it may not be possible to classify an animal's genotype solely on the basis of a phenotype. In these more ambiguous cases, genetic testing will play a vital role in the unequivocal determination of genotypes, allowing appropriate and informed breeding decisions.

Chromosomal Abnormalities

Occasionally, problems arise during mitosis, meiosis, or fertiliza­tion, resulting in chromosomal, or karyotype, abnormalities such as a chromosome missing from a pair (monosomy) or an extra chromosome (trisomy). Many karyotypic abnormalities have been identified in all large animal species, but not every chromosomal abnormality has been associated with an overt disease phenotype.^11,12 Some examples of common chromosomal abnormalities include the absence of an X chromosome, called XO Turner syndrome, which results in female infertility. An additional X chromosome in XXY Klinefelter syndrome causes underdeveloped males with poorly developed male sexual behavior. In addition to abnormal numbers of sex chromosomes, rare trisomies of autosomal chromosomes have been observed in mammals; however, monosomies of autosomes are generally not compatible with life in mammals. An additional chromosomal abnormality can occur when two chromosomes unite into a single large chromosome, called a Robertsonian translocation, or centric-fusion translocation, leading to reduced reproduc­tive capacity in some species and early embryonic death in cattle. Because of possible chromosomal abnormalities, several countries have instituted mandatory karyotype examination of some breeding cattle. In addition, clinicians should consider karyotyping any animal that presents for reduced fertility or has multiple abnormalities.

Breeding Schemes

As a result of numerous advances in the field of genetics, the genetic causes of many diseases and traits are known and can be tested before using an animal in a breeding program. As research continues, more DNA-based tests are expected; however, clients may be interested in testing for a disease or trait that does not yet have a known genetic cause. For these situations, sires can be tested using breeding trials, as described in Table 51.1. For example, if a sire is bred to carrier females for a disease of concern, he would have to produce 10 normal offspring to ascertain that he is not a carrier with 95% confidence, 16 normal offspring to have 99% confidence, and 24 normal offspring to have 99.9% confidence that he is not a carrier for the disease of concern. The time required to complete test mating can be significantly shortened using superovulated affected females, insemination, and embryo transfer (two per recipient) followed by early cesarean section at 60 days and examination of the offspring for the genetic defect(s) of concern.

■ TABLE 51.1

Test Mating Schemes to Examine Males for Genetic Traits or Diseases at Various Confidence Levels

Normal Offspring Needed to Reach Probability Level of:

^aTests for only one trait.

^bTests for all undesirable recessive traits.

It has historically been common practice for artificial insemination (AI) studs to conduct such test matings for reces­sive genetic diseases. However, now that many of the deleterious mutations are known, genetic testing, rather than test mating, is the current preferred approach. As mentioned previously, the dairy and beef cattle, swine, and sheep industries are aggressively pursuing whole-genome genetic testing, often in combination with offspring data. Test matings are thus largely relegated to the arena of historical interest, although there may be unique situations or other species in which a test mating may still be a legitimate approach.

Positive and Negative Selection

Breeders can improve their breeding stock by selecting for or against specific traits and diseases. These processes are known as positive and negative selection, respectively. A breeder may choose to breed an animal because it has desirable genes or qualities (positive selection). Alternatively, someone may choose not to breed an animal that has been shown, through either genetic or breeding tests, to carry an undesirable trait or disease (negative selection). Ultimately, both scenarios will achieve the goal of improved breeding stock.

Recommendations for Breeding Programs

■ BOX 51.1

Genetic Counseling for Single-Gene Autosomal Recessive Diseases

From the standpoint of conserving as much genetic diversity as possible within the closed gene pool of registered purebred animals, regardless of species, it is recommended by veterinary geneticists to retain heterozygotes (carriers) of autosomal recessive diseases as breeding animals, assuming they are otherwise good breeding candidates and the disease has a known, genetically testable mutation. If the animal has many other desirable traits, the genes underlying those traits can still be retained in the gene pool by breeding the carrier individual only to homozygous normal (clear) animals for the recessive disease in question. All offspring will be either homozygous normal or, at worst, carriers; no affected offspring will be produced. All offspring should be genetically tested for the gene in question. Exceptional offspring (again, regardless of carrier status) should be retained and used for breeding. Only slowly, and over several generations, using genetic testing, should the carriers be removed from the gene pool. Consider the example of an autosomal recessive hydrocephalus in Friesian horses; this disorder has been associated with a nonsense mutation in B3GALNT2, where homozygotes for the affected allele exhibit hydrocephalus, while heterozygotes (carriers) are normal. The frequency of heterozygous individuals in the Friesian breed is ~17%. 3 Immediate removal of 17% of the gene pool, based on carrier status of this one gene, could create cata­strophic new genetic bottlenecks. The recommendation in this scenario is to continue breeding heterozygotes, but only to homozygous-clear mates, therefore producing no foals with hydrocephalus but maintaining as much genetic diversity in the breed as possible. Ultimately, veterinary genetic counseling needs to consider which individuals should be bred and to whom they should be mated, keeping both short-term (individual offspring of this mating) and long-term (genetic health of the breed) perspectives. Finally, note that this recommendation is not appropriate in dominant genetic mutation scenarios; heterozygous individuals with dominant mutations are consid­ered affected and typically should not be bred, depending on the severity of the condition.

in the herd and overall breeding population is important in order to avoid inbreeding depression and the increase of recessive diseases. In some industries, such as swine, where crossbreeding is common, this is less of a concern. In other industries, such as dairy cattle, producers are increasing their utilization of crossbreeding over and above the historical standard. Most commercial AI studs and/or breed association mating programs in the dairy industry are now designed with an option to limit inbreeding, typically accomplished with simple pedigree analysis but increasingly using genomic data; however, the producer must choose to apply that option. Breed associations may eventually need to intervene by actively soliciting and providing incentives for outbred animals. Produc­ers, as well as the commercial stud owners, must carefully balance the short-term gains that may come from inbreeding with the long-term consequences of the overall narrowing of the gene pool.

Obtaining Genetic Information

Because of the ever-increasing wealth of genetic information, the most complete and current information can be found using online resources. There are now hundreds of recognized single-gene traits and diseases across all large animal species; descriptions of each one is beyond the scope of this chapter.

■ BOX 51.2

Recommended Websites for Large Animal Genetics

OMIA-Online Mendelian Inheritance in Animals: horse, cattle, sheep, goat, pig, various other species http://omia.angis.org.au/

U.S. Department of Agriculture, Agriculture Research Service, Animal Genomics and Improvement Laboratory: dairy cattle, dairy goat http://aipl.arsusda.gov/

Veterinary Genetics Laboratory at the University of California, Davis: horse, cattle, sheep, goat, pig, various other species http://www.vgl.ucdavis.edu/services/index.php Because it is difficult for the practitioner to be familiar with all of them, online resources become vital. It is recommended that clinicians use websites produced by reputable institutions to obtain the most accurate and referenced information available (Box 51.2). Alternatively, clinicians are encouraged to contact the Veterinary Genetics services available at many veterinary schools if they are unable to locate the information they require.

REFERENCES

The complete list of references can be found at www.expertconsult.com.

REFERENCES

1. Beever JE, Smit MA, Meyers SN, et al: A single-base change in the tyrosine kinase II domain of ovine FGFR3 causes hereditary chondrodysplasia in sheep, Anim Genet 37:66, 2006.

2. Passos DT, Rodrigues EE, Rodrigues NC, et al: Allele frequency of the spider lamb syndrome in Brazilian Hampshire Down and Suffolk flocks, SmallRumin Res 83:79, 2009.

3. Beck CL, Fahlke C, George AL: Molecular basis for decreased muscle chloride conductance in the myotonic goat, Proc NatlAcad Sci USA 93:11248, 1996.

4. Naylor JM, Nickel DD, Trimino G, et al: Hyperkalaemic periodic paralysis in homozygous and heterozygous horses: a co-dominant genetic condition, Equine Vet J 31:153, 1999.

5. Rudolph JA, Spier SJ, Byrns G, et al: Periodic paralysis in Quarter Horses: a sodium channel mutation disseminated by selective breeding, Nat Genet 2:144, 1992.

6. Khatkar MS, Thomson PC, Tammen I, et al: Quantitative trait loci mapping in dairy cattle: review and meta-analysis, Genet Sel Evol 36:163, 2004.

7. Tenghe AMM, Bouwman AC, Berglund B, et al: Genome-wide association study for endocrine fertility traits using single nucleotide polymorphism arrays and sequence variants in dairy cattle, J Dairy Sci 99:5470, 2016.

8. Pryce JE, Hayes BJ, Bolormaa S, et al: Polymorphic regions affecting human height also control stature in cattle, Genetics 187:981, 2011.

9. Bolormaa S, Pryce JE, Kemper K, et al: Accuracy of prediction of genomic breeding values for residual feed intake and carcass and meat quality traits in Bos taurus, Bos indicus, and composite beef cattle, J Anim Sci 91:3088, 2013.

10. Fleming A, Abdalla EA, Maltecca C, et al: Invited review: reproductive and genomic technologies to optimize breeding strategies for genetic progress in dairy cattle, Arch Anim Breed 61:43, 2018.

11. McFeely RA: Chromosome abnormalities, Vet Clin North Am Food Anim Pract 9:11, 1993.

12. Nicholas FW: Introduction to veterinary genetics, ed 2, Oxford, England, 2003, Blackwell, p 93.

13. Ducro BJ, Schurink A, Bastiaansen JWM, et al: A nonsense mutation in B3GALNT2 is concordant with hydrocephalus in friesian horses, BMC Genomics 16:761, 2015.

14. 2018 American Quarter Horse Association Official Handbook of Rules and Regulations, 66^th ed. Available at: https://www.aqha.com/media/24175/ aqha-rulebook-2018.pdf. (Accessed 22 April 2018).