New Background - The South African Veterinary Associationsava.co.za/wp-content/uploads/2015/10/genetic-counseling... · Web view2018. 10. 12. · Disorders for which the mode of

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Genetic counselling: Educating the small animal practitioner in

South AfricaDe Cramer K.G.M.1 and Fraser M.F.2

1Rant en Dal Animal Hospital, Mogale city, South Africa, [email protected] 2Inqaba Biotechnical Industries (Pty) Ltd, Hatfield, Tshwane, South Africa, [email protected]

1 BACKGROUND

Responsible dog breeders are those that make the welfare of each animal and its offspring their first priority. These breeders are those that participate in genetic health improvement schemes such as hip and elbow improvement schemes and test their dogs to detect carriers of known genetic disorders. It is unfortunate that the negative publicity which unethical breeders engender, tarnishes the credibility of a legion of breeders who do a splendid job. Unfortunately, a rift has developed between breeders and the veterinary profession. This stems from a number of factors involving the veterinarian, breeder and pet owner triad. Following the diagnosis of a known genetic disorder (or often a suspected genetic disorder) in their beloved pet, the pet owner is naturally disgruntled. Such pet owners frequently lash out by claims of indiscriminate breeding, inbreeding and intentional breeding of carriers of genetic disorders with perverse financial intent. Pet owners and their veterinary surgeons often fail to understand that with many genetic disorders, it is not realistic to expect breeders to predict and prevent all genetic disorders. A more realistic expectation is to diminish the prevalence of genetic disorders using the tools available to them. Prevention of producing puppies free of genetic disorders can only be achieved for those few disorders for which there is a genetic test available to breeders. Breeders that have tried their utmost are then left to defend themselves against unfair claims and feel despondent. These breeders also claim that many veterinarians are not adequately knowledgeable to address such unfair claims and are poorly educated in the field of Canine and Feline genetics to defend them. Unfortunately, breeders that do not participate in health improvement schemes, also use this as a convenient excuse to continue not participating. Their claim is that they will be criticised whether they try or not. Criticism is only justified against those that make little or no effort in pursuing genetic health in their breeding stock and not against those that do. Progressive responsible breeders of purebred dogs and cats count on the veterinary profession to defend them and counsel them in their quest towards genetic progress. The purpose of this manuscript is to familiarise the small animal practitioner with the basic knowledge and tools to achieve this goal and to empower veterinarians to actively promote participation by breeders in health improvement schemes. Everyone stands to gain from these efforts, not least the pet.

Breed registering bodies and authorities are the key stake holders that may influence genetic health of a breed (Crispin 2011). It may be argued that the individual breed “parent bodies” of the respective breeds are the custodians of the individual breeds and have the largest impact on breed health as they dictate breed standards directly or indirectly for others all over the world. There is an ongoing onslaught against some of these bodies because of claims that their breed standards blatantly oppose health (all be it unintentional), rather than promote it

mailto:[email protected]

mailto:[email protected]

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(Asher et al. 2009). The reluctance to change these breed standards stems from the fact that changing features in dogs may threaten the very existence of the breed in its current form. Breeding strategies that involve judging on the production of over-exaggerated conformational features have attracted severe criticism (Meyers‐Wallen 2003; Crispin 2011). Credit is due to those that courageously support those individual breeders who have suggested drastic changes to breed standards which negatively affect the health and wellbeing of the breeds concerned. Unfortunately, mostly such efforts fail, and small animal practitioners will continue to attend to many breeds related conformational defects until hopefully a major global mind shift regarding these issues emerges. The example of the English Bulldog and French Bulldog need special mention in this regard. These breeds are plagued with brachycephalic obstructive airway syndrome (Ladlow et al. 2018) and a high incidence of caesarean sections (Munnich and Kuchenmeister 2009). A recent study showed that brachycephalic obstructive airway syndrome risk increases sharply in a non-linear manner as relative muzzle length shortens (Packer et al. 2015). When dogs with these extreme phenotypes are delivered by caesarean section, there is no motivation for selection against this aesthetic feature and hence the associated morbidity and mortality will increase. A recent study was also undertaken to solve the problem of emergency caesarean sections in the English Bulldog by devising a protocol whereby a preparturient caesarean section is performed in a significant proportion of high risk obstetric populations (De Cramer 2017). Although the latter study mitigates the problem of the emergency caesarean section in affected breeds it does nothing to advance the wellbeing of the breed as a whole and only deals with the clinical existing problem of dystocia in affected breeds. When dogs with these extreme phenotypes are delivered by caesarean section, there is no motivation for selection against this aesthetic and hence the associated morbidity and mortality will increase. Given the balance of evidence, it is fair to conclude that brachycephaly is the underlying main contributor to these two problems and that selection against brachycephaly is more likely to render favourable results than selecting against the prevalence of its secondary component, the caesarean section itself or selection against brachycephalic obstructive airway syndrome. Selection for healthier conformations should therefore be our first aim (Packer et al. 2015; Ladlow et al. 2018). It has been suggested that for the maximum wellbeing of future generations, we should abandon the most predisposed breeds (those with the poorest health record and highest number of inherited disorders) while loosening the genetic barriers between the remaining breeds to promote genetic variability (Jeppsson 2014).

A recent study found that 48% of companion animal veterinarians were advising clients against purchasing a pedigree dog breed due to inherited disorders (Farrow et al. 2014). However, it important to recognise that not all deleterious traits should be attributed to breed standards (Summers et al. 2010). Neither can it be assumed that only purebred dogs are afflicted with deleterious genes. Prevalence of genetic disorders in both purebred and mixed-breed dog populations is related to the specific disorder (Bellumori et al. 2013). Unfortunately, fashion (social influence) are more important than function in determining the popularity of dog breeds (Ghirlanda et al. 2013). A new fad that has entered the dog arena is the designer breeds and has become increasingly popular in some parts of the world and getting foothold locally as well. The labradoodle (Labrador Retriever + Standard Poodle) is best known. There is a common misconception that because the designer breeds originated from founders of distinct breeds, they will automatically be healthier and less prone to inherited disorders. Undoubtedly outcrossing will increase heterozygosity and reduce the

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frequency of disease-causing alleles in a breed. However, the labradoodle as a “new breed” can only be healthier than their founding stock in the long term if they originated from lineages of the founding stock known to have a lower prevalence of genetic diseases than their respective breed averages. Designer breeds are however likely to suffer from all conditions prevalent in the founding breeds it originated from, posing more challenges to eliminate more disorders.

Breeders typically select superior stock based on phenotypic breed characteristic and performance in show rings. It is easy for breeders to select for such traits as they are visually evident in their breeding stock. Selecting for genetic health is thwarted by the fact that genetic disorders may not always be present in the parent stock, skip generations because of carrier states, complex modes of inheritance and because many genetic disorders may manifest themselves much later on in life, out of the eye of the breeder. Breeders also often pride themselves in holding the notion that good selection is mainly based on breeder experience and a keen eye. Veterinarians should remind them that this is true only for phenotypic traits but that true genetic progress and selection for genotype can only be achieved by the collective efforts by many breeders involving a significant proportion of the breeding population (Hedhammar et al. 2011). More specifically, these efforts involve participation in health improvement schemes, genetic testing, respecting inbreeding coefficient limits, maintaining genetic diversity and the establishment of health registries.

2 STATUS QUO IN SOUTH AFRICA

The USA has the largest number of registered puppies with numbers approaching one million per year and some others are UK 250 000, and Japan 350 000. South Africa is vastly outnumbered by these statistics. For 2016, the total number of registered puppies was ≈ 22 000. The top twelve breeds were; Boerboel 4222 (South African Boerboel Breeder 2016), GSD ≈1300 (SA 2016), English bulldog 1278, Labrador Retriever 1144, Rottweiler 1140, Staffordshire Bull Terrier 908, Bull Terrier 855, Golden Retriever 661, Miniature Schnauzer 586, Pomeranian 564, Beagle 389, and Yorkshire Terrier 336 (Kennel Union Of South 2016). Given these statistics, it is no surprise that we have a limited role to play in funding research towards developing tests and genetic progress in general. Because hip dysplasia is a serious orthopaedic condition causing major discomfort to the pet, leading to huge expenses to pet owners (Malm et al. 2010) and because it is a very common problem in many local breeds (Kirberger 2017), it deserves special attention. Despite being a proven major problem, only seven breeds have breeding restrictions for hip dysplasia in South Africa (KUSA). A recent local study on prevalence of hip dysplasia in Rottweilers (do have breeding restrictions for hip dysplasia) and Labrador Retrievers (do not have breeding restrictions for hip dysplasia), proved that prescribing minimum breeding requirements significantly improved the breeding stock (Kirberger 2017). Factors that impact voluntary participation by breeders to health improvement schemes need to be addressed. Dog registries depend on puppy registration fees for their survival and cannot impose restrictions in the absence of significant support from their members for such proposal. Some breed authorities claim that by imposing forced breeding restrictions tarnishes the breeds’ reputation by focussing the attention of the public on the assumption that their breed must be riddled with problems. This they use as an excuse to continue not to participate. For those that do participate it is claimed that no good deed goes unpunished. The GSD federation of South Africa is given as an example. They claim

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that despite imposing breeding restrictions on hip dysplasia for decades, some members of the veterinary profession continue to perceive and portray the German Shepherd Dog breed as one of the worst affected breeds (SA 2016). This they claim is very unfair given that the German Shepherd Dog breed ranks number 37 on the AKC list of worst affected dog breeds regarding HD (AKC). Against this background the German Shepherd Dog Federation of South Africa is reluctant to impose breeding restrictions on elbow dysplasia in their breed.

Misconceptions regarding the breeding recommendations issued by breeding authorities on breeding with slightly affected dogs, also exist. Some members of the public and veterinary profession hold the view that the breeding restrictions imposed are indeed too slack and that this explains the continued prevalence of hip dysplasia. This notion exists despite irrefutable published proof that although hip dysplasia improvement schemes leads to significant decreases in prevalence it cannot be expected to totally eradicate hip dysplasia (Mäki 2004; Ginja et al. 2010). This is true for all polygenetic traits and for traits influenced by environmental factors. As veterinarians we should also be aware that it is widely accepted to breed with known carriers en-route to improvement. This statement requires clarification. Again, taking hip dysplasia as an example. On the AKC list for worst affected breeds the percentage of dogs within the breed considered to have excellent hips ranged from 0.3% to 55% with most being under 10%. If one were to allow only these excellent dogs as breeding candidates, 90% of the breeding population would be excluded and lost forever. This would destroy the breed altogether by diminishing genetic diversity. Furthermore, given the mode of inheritance of hip dysplasia, it is unlikely that such purist approach would significantly improve matters when compared to the current approach (Mäki 2004). It is more likely that due to the genetic bottle neck that such approach would create, it would be impossible to select for superior phenotypic traits whilst at the same time also selecting against a multitude of other genetic disorders (Leroy 2011). Unbeknown to many within the veterinary profession, against the background of the threat of diminishing genetic diversity, it is also scientifically sound to allow the breeding of known carriers of recessive genes subject to the conditions described in this manuscript.

3 ESTABLISHMENT OF OPEN ACCESS HEALTH DATA BASE REGISTERS

An open health data base register is a register of individuals that have been tested for a specific genetic disorder and the results are made available to the public online There exists no doubt that the establishment of open access health data base registers are powerful tools enabling breeders to select least affected lineages (Hedhammar et al. 2011). The question remains whether such registers should be an open health data base register, and should this be enforced or not? The obvious advantage is that this helps breeders in planning mating’s accordingly and it allows prospective pet owners reducing risk. In some instances, making public results of tests is made obligatory by a breeding society and in others it is voluntary and in others it is semi open meaning that it only reports on individuals with positive results or only reports results when the breeder wishes to do so. Although ideal, obligating the display of all results to the public may act as a hindrance of breeders to participate to genetic improvement schemes. This is because of fear of stigmatisation and the negative impact negative results may have on their reputation as breeder. Also, when breeders have no idea about the prevalence of a specific mutant allele in its gene, many breeders opt not to make public their results initially. However, once these breeders have a grip on the situation and

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have established clear status of individuals, they are less reluctant to make their results public. All parties involved, breeders, veterinarians and breeding authorities should be open minded about this and consider respecting the wish for anonymity. Albeit that the concept of non-disclosure to the public is not ideal, any measure that ultimately leads to increasing the percentage of animals tested within a population, should be strongly supported. Genetic progress depends on the number of breeders participating. It is fair to assume that breeders that pay good money to have tests performed, may have at least the good intention to retire affected individuals and carriers. It is in the interest of the genetic welfare of our purebred pet population that breeders be allowed to do so anonymously and in dignity if they so wish. Participation to a health improvement scheme should be the first priority whether perfect or not. Such improvement schemes can then later “upgrade” to a voluntary, semi-open or open health register. Similar approaches have been followed abroad.

4 THE GENETIC COUNSELLING PROCESSES

Veterinarians have an ethical responsibility to promote genetic welfare of the pet population. If a genetic test is available, it should be utilized by breeders so that pet owners are not burdened with predictable genetic disease. To protect their clients, veterinarians should promote the purchasing of dogs from breeders that are actively involved in health improvement programmes. In cases where breeders do not test for genetic disorders, it is a personal decision whether the pet owner wishes to test or not. Before the owner has the test done, they might want to discuss with their veterinarian what they wish to do if their pet has a high likelihood of developing a genetic disorder later in life. It is important to note that it may be counterproductive for breeding authorities to make all available health tests mandatory for their breeder members. This would risk alienation of breeders from organizations that regulate them - a step by step approach may be more accepted. Due to; the very many known and suspected genetic disorders in both dogs (≈700) and cats (≈350) (sources, CIDD, IDID, OMIA online databases), the fact that some conditions are very rare and that not all breeds are well represented everywhere, all veterinarians cannot be expected to be familiar with all the known or suspected genetic conditions. However, veterinarians can make sensible contributions if they are knowledgeable about the common specific disorders occurring in breeds regularly seen by them. This will assist them not only in genetic counselling but also in the differential diagnosis of disease in the common breeds they attend to. In addition, they should either know the screening tests available or have access to the list of such genetic conditions for which tests are available. Sterling work has already been completed by some of the larger dog and cat registering authorities abroad who have gone to great lengths to formulate breed specific breeding recommendations that is accessible to the public and veterinary profession. Three excellent online resources for information on canine genetic disorders databases are the Canine Inherited Disorders Database (CIDD), the Inherited Diseases in Dogs (IDID) resource, and NIH’s Online Mendelian Inheritance in Animals (OMIA) database (Nicholas et al. 2011).

The first step in the genetic counselling process is identifying the genetic disorders relevant to the breed and weighting them for the individual breeder (Hedhammar et al. 2011). The genetic counselling should include only recommendations on genetic disorders that really matter to the breeders in real life. Such breed specific lists are already available on many popular breed specific websites, but the list of conditions can be weighted differently

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according to meet the needs of individual breeders. Whether a specific genetic disorder poses high risk or low risk depends on the severity of the impact that the genetic disorder has on the dog or cat, the prevalence of the disorder in the breeding population under investigation, heritability of the trait, mode of inheritance of the genetic disorder, whether phenotypic or genotypic test is available and whether there is a health improvement scheme in place, are all important in this decision-making process.

South Africa’s own breed, the Boerboel, is used as an example. Most Boerboel breeders will agree that; hip dysplasia, elbow dysplasia, temperament, entropion, heart disease, wobbler like syndrome, susceptibility to demodectic mange, formation of excessive calluses over pressure points, pyoderma, vaginal hyperplasia, umbilical hernias, cryptorchidism and male subfertility should appear on the problem list. The first three traits are likely to appear on everyone’s list in the same order of importance and personal experience will dictate the order of the others. The genetic health of the subpopulation of the breeder of that breed, will then dictate against how many of the listed disorders they can afford to select against simultaneously. Based on impact on the pet and its owner, strict selection against hip dysplasia, elbow dysplasia and temperament, should be considered non-negotiable. Fortunately, this is supported by many but not all. Boerboel breeders that are fortunate enough to have achieved significant individual progress regarding these traits by having consistently better scores than the breed average may then progress to select against other traits on the list or weight them differently based on need. Harsh criticism by some against failure to select against vaginal fold hyperplasia by some breeders may not be justified. This is because, in the bigger picture, the Boerboel breed has many major hurdles to negotiate regarding very many other more serious problems that have major impact on health before they attempt to address the minor ones. For instance, hip and elbow dysplasia has devastating effects on the pet often resulting in euthanasia, costly surgery and lifelong medical treatment with essentially life long suffering (Malm et al. 2010). In contrast, vaginal fold hyperplasia can easily be resolved by simple sterilisation and has no lifelong implications. There are many other such examples.

Domestication is directly associated with the accumulation of genetic diseases due to the significant selection pressures applied on the population. This is directly observed when comparing different animal species and the number of genetic diseases associated (Figure 1), while domesticated animals such as dogs, cattle and cats have >700, >500 and >300 genetic diseases identified respectively, wild animals like the Gray wolf, cheetah, chimpanzee and springbok have 6, 5, 4 and 4 genetic diseases identified. Similarly, the animal with the most characterised genetic diseases is the dog, which is attributed to the parallel evolutionary pressure on the dog and human as they evolved together over 35’000 years (Wang et al. 2013). While there are >700 genetic diseases estimated in dogs, only 243 have been genetically characterised (OMIA: Figure 1). This changes our perspective on genetic disease in the dog as there are still numerous conditions in dogs that have never been seen, have no name or no published work. Therefore, it is advised that suspicion around heritability should be raised when they are clearly over-represented in a breed population or the same disease is observed within members of a family or lineage.

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Figure 1. The number of genetic diseases identified across species (Dog, Cow, Cat, Sheep, Pig, Chicken, Horse, Goat, Rabbit, Deer, Gray Wolf, Cheetah, Springbok and Chimpanzee), OMIA as of 31 July 2018.

dog cattle cat horse chicken goat gray wolf cheetah chimpanzee springbok

-50

50

150

250

350

450

550

650

750719

523

341

233219

83

6 5 4 4

243

148

6244 44

11 1 2 1 0

420

204 208

129

48 36

1 2 2 4

Total Genetic Diseases Genetic Mutations Characterised Potential Models for Human Disease

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5 MODE OF INHERITANCE OF GENETIC DISORDERS

Veterinarians do not need to be professional geneticists to develop a fundamental understanding of the effects of molecular genetics on veterinary medicine. However, basic knowledge of the mode of inheritance of a trait is essential to the control of genetic diseases. The terms dominant versus recessive and autosomal versus sex-linked requires explanation.

With the exception of genes on the X and Y chromosomes (i.e., the sex chromosomes), genes come in pairs. Non-sex chromosomes are called autosomes and the paired genes on them are termed autosomal genes. Diseases caused by mutations in autosomal genes are classified according to whether one or two copies of the mutant gene are needed to produce disease. If only one copy of a mutant gene is needed to produce the disease and the other copy of the gene is normal, the resultant disease is called autosomal dominant. If both copies of the gene must be mutant to cause disease, the term autosomal recessive is used.

Dominant disorders tend to be less troublesome to breeders than are recessive disorders because often the disorder is detected before an animal is bred. This is true for only those disorders that manifest early in life before the normal breeding age. Disorders that have a late onset can still be problematic to detect timeously.

Autosomal recessive genes are entirely different. Identifying carrier animals usually is not possible until mating of two previously unknown carriers produces one or more affected offspring. By this time, the animal has already been bred and when undiagnosed carriers are mated to non-carriers, 50% of their offspring will carry the disease gene without anyone knowing.

5.1 Autosomal disorders

Autosomal disorders are those that are caused by a mutant allele on any of the chromosomes other than the sex chromosomes. Autosomal disorders may be recessive or dominant and are respectively known as autosomal recessive or autosomal dominant disorders.

5.1.1Autosomal recessive (also known as simple recessive) mode of inheritance

In autosomal recessive inheritance, two copies of an abnormal gene must be present in order for the disease or trait to develop. The parents of an individual with an autosomal recessive condition each carry one copy of the mutated gene, but they typically do not show signs and symptoms of the condition. Autosomal recessive disorders are typically not seen in every generation of an affected family. Unless there is a test available, carriers of recessive traits cannot be detected except by the production of affected offspring. Only individuals homozygous for the detrimental mutations will manifest the disorder phenotypically. Therefore, parents may be unaffected but if both are carriers, there is a 25% probability of an individual being affected, 25% of being clear and 50% probability of being a carrier. It has to be remembered that the percentage probability is the probability per fertilisation and refers to an average over many fertilizations. Breeders that fail to understand this, may argue the point that when they have crossed a carrier to a clear and they have a litter of four, they only expect

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one carrier. But reality is that anything from zero to four carriers is a statistical probability for this four-puppy litter.

If only one parent is a carrier, no affected offspring can be produced but offspring may be carriers also.

5.1.2Autosomal dominant disorders

Autosomal dominant disorders only require one copy of the mutation to be present for the disease or disorder to manifest. Thus, an individual could present with the disease if it is heterozygous or homozygous for the mutation. Furthermore, carrier states reveal themselves and this allows for easy selection against the disorder and thus are far less common.

5.2 Sex linked disorders

Disorders may be sex (gender) linked because the mutant allele is found on either the X or Y chromosome. Depending on whether the mutation is on the X or Y chromosome the disorder is termed X-linked or Y-linked. Y-linked disorders are very rare. X-linked disorders may further be complicated by incomplete penetrance as described below

5.2.1X-linked dominant disorders

X-linked dominant disorders are caused by mutations in genes on the X chromosome, one of the two sex chromosomes in each cell. In females (who have two X chromosomes), a mutation in one of the two copies of the gene in each cell is sufficient to cause the disorder. In males (who have only one X chromosome), a mutation in the only copy of the gene in each cell causes the disorder. A characteristic of X-linked inheritance is that the sire cannot pass X-linked traits to their male offspring (thus no male-to-male transmission). Some X-linked dominant disorders are lethal in hemizygous males (e.g. Samoyed hereditary glomerulopathy) (Jansen et al. 1986). This means that only heterozygous females are normally present in the population as most affected males die from renal failure before age 15 months. A homozygous female is expected to succumb to the same timeline. In a heterozygous female however, disease progression is much slower and less severe.

5.2.2X-linked recessive disorders

X-linked recessive disorders are also caused by mutations in genes on the X chromosome. In males (who have only one X chromosome), one altered copy of the gene in each cell is sufficient to cause the condition. Therefore, X-linked recessive disorders are biased in the male population because heterozygous females do not present with the disease but will present in a male dog as he only has one X chromosome. Because it is unlikely that females will have two altered copies of this gene, males are affected by X-linked recessive disorders much more frequently than females.

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Table of common autosomal recessive and autosomal dominant heritable diseases for which tests are available in domestic felids

Breed Disease Gene Onset AR

AD Reference

Abyssinian & Somali breeds

Rod cone dysplasia CRX Early X (Menotti-Raymond et al. 2010)

African Black-Footed Cat Progressive retinal atrophy IQCB1 Early X (Oh et al. 2017)Bengal Progressive retinal atrophy Early X (Ofri et al. 2015)Burmese Hypokalaemia WNK4 Early X (Gandolfi et al. 2012)Burmese GM2 gangliosidoses HEXB Early X (Bradbury et al. 2009)Main Coon Spinal muscular atrophy LIX1 Early X (Fyfe et al. 2006).Maine Coon Hypertrophic Cardiomyopathy MYBPC3 Early-

Late X (Meurs et al. 2005)

Norwegian forest cats Glycogen storage disease type IV GBE1 Early X (Fyfe et al. 2007)Persian Progressive retinal atrophy Early X (Alhaddad et al. 2014)Ragdoll Hypertrophic Cardiomyopathy MYBPC3 Early-

Late X (Meurs et al. 2007)

Various Pyruvate kinase deficiency PKLR Early-Late

X (Grahn et al. 2012)

Various Polycystic kidney disease PKD1 Early-Late

X (Lyons et al. 2004)

Various Retinal degeneration CEP290 Late X (Menotti-Raymond et al. 2007)

*AR – Autosomal Recessive Mode of Inheritance*AD – Autosomal Dominant Mode of Inheritance

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Table of autosomal recessive and autosomal dominant heritable diseases for which genetic tests are available in domestic canines

Breed Disease Gene Onset AR AD XL ReferenceMastiff breeds Progressive retinal atrophy RHO Early X (Kijas et al. 2002)Basenji Progressive retinal atrophy S-antigen Late X (Goldstein et al. 2013)Dachshunds, Spaniels & Curley Coated Retriever

Progressive retinal atrophy (cord1) RPGRIP1 Early X (Mellersh et al. 2006a)

Wirehaired dachshund Progressive retinal atrophy (cord2) NPHP4 Early X (Palánová et al. 2014)Golden Retriever Progressive retinal atrophy (GR1) SLC4A3 Late X (Downs et al. 2014b)Golden Retriever Progressive retinal atrophy (GR2) TTC8 Mid-

LateX (Downs et al. 2011)

Various Progressive retinal atrophy (prcd)β PRCD Mid-Late

X (Zangerl et al. 2006)

Irish Setters Progressive retinal atrophy PDE6B Early X (Suber et al. 1993)Shetland Sheepdogs Progressive retinal atrophy CNGA1 Mid X (Wiik et al. 2015)Samoyed & Siberian Husky

Progressive retinal atrophy RPGR Mid X (Zhang et al 2002)

Collie breeds Progressive retinal atrophy RD3 Early X (Kukekova et al. 2009)Corgi, Pomeranian & Chinese Crested

Progressive retinal atrophy PDE6A Early X (Petersen‐Jones and Entz 2002)

Various Progressive retinal atrophy C2orf71 Late X (Downs et al. 2014a)English Bull Terriers Polycystic kidney disease PKD1 Early X (Gharahkhani et al. 2011)Various Cone degeneration CNGB3 Early X (Yeh et al 2013)Various Collie eye anomaly NJEH1 Early X (Parker et al. 2007)Various Canine multifocal retinopathy VMD2 Early X (Guziewicz et al. 2007)Labrador Retriever Centronuclear myopathy PTPLA Early X (Pelé et al. 2005)Great Dane Centronuclear myopathy BIN1 Early X (Böhm et al. 2013)Golden Retriever Muscular dystrophy DND Early X (de Lima et al. 2007)Bedlington Terriers Copper Toxicosis COMMD1 Early-

LateX (Forman et al. 2005)

Various Cystinuria SLC31 Early X (Henthorn et al. 2000)Various Degenerative myelopathy SOD1 Mid X (Zeng et al. 2014)Various Factor VII deficiency EGF-2 Early X (Callan et al 2006)German Shepherd Dog & White Shepherd Dog

Factor VIII deficiency Factor VIII Early X (Christopherson et al. 2014)

Australian & American Shepherd breeds

Hereditary cataract HSF4 Early-Late

X (Mellersh et al. 2006a)

Staffordshire Bull Terriers, Hereditary cataract HSF4 Early X (Mellersh et al. 2006b)

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Breed Disease Gene Onset AR AD XL ReferenceBoston Terrier & French BulldogVarious Hyperuricosuria SLC2A9 Early X (Bannasch et al. 2008)Golden Retriever Ichthyosis PNPLA1 Early X (Grall et al. 2012)Beagle & Border Collie Imerslund-Grasbeck Syndrome CUBN Early X (Drögemüller et al. 2014)Rottweiler & Black Russian Terrier

Juvenile Laryngeal Paralysis and Polyneuropathy

RAB3GAP1 Early X (Mhlanga-Mutangadura et al. 2016; Mhlanga‐Mutangadura et al. 2016)

Staffordshire Bull Terriers L-2-Hydroxyglutaric aciduria L2HGDH Early X (Penderis et al. 2007)Russel Terriers Late Onset Ataxia CAPN1 Early X (Forman et al. 2013)Various Lafora disease NHLRC1 Mid-

LateX (Hajek et al. 2016)

Miniature Schnauzer & Russel Terriers

Congenital Myotonia CIC1 Late X (Rhodes et al. 1999)

Various Multi-Drug Resistance MDR1 Early X (Mealey et al. 2001)Beagle Musladin-Leuke Syndrome ADAMTSL2 Early X (Bader et al. 2010)Rhodesian Ridgeback Myoclonic epilepsy DIRAS1 Early X (Wielaendera et al 2017)Beagles Cerebellar abiotrophy SPTBN2 Early X (Forman et al. 2012)Vizsla Neonatal cerebellar cortical degeneration SNX14 Early-

MidX (Fenn et al. 2016)

Chihuahua Neuronal ceroid lipofuscinosis MFSD8 Early-Late

X (Ashwini et al. 2016)

Dachshund Neuronal ceroid lipofuscinosis TPP1 Early X (Awano et al. 2006a)American Bulldog Neuronal ceroid lipofuscinosis CTSD Early X (Awano et al. 2006b)Tibetan Terrier Neuronal ceroid lipofuscinosis ATP13A2 Mid-

LateX (Farias et al. 2011)

Golden Retriever Neuronal ceroid lipofuscinosis CLN5 X (Gilliam et al. 2015)Border Collie & Australian Cattle Dog

Neuronal ceroid lipofuscinosis CLN5 Early X (Melville et al. 2005)

Australian Shepherd Neuronal ceroid lipofuscinosis CLN6 Early X (Katz et al. 2010)Chinese Crested Neuronal ceroid lipofuscinosis MFSD8 Early X (Guo et al. 2014b)Australian Shepherd Neuronal ceroid lipofuscinosis CLN8 Early X (Guo et al. 2014a)Setter Neuronal ceroid lipofuscinosis CLN8 Early X (Katz et al. 2005)Dachshund Neuronal ceroid lipofuscinosis PPT1 Early X (Sanders et al. 2010)American Staffordshire & Pit Bull Terriers

Neuronal ceroid lipofuscinosis ARSG Early X (Abitbol et al. 2010)

Standard Poodle Neonatal encephalopathy ATF2 Early X (Chen et al. 2008)Various Phosphofructokinase deficiency PFK Early X (Smith et al. 1996)

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Breed Disease Gene Onset AR AD XL ReferenceBeagle, Labrador Retriever & Pug

Pyruvate kinase deficiency PKLR Late X (Inal Gultekin et al. 2012)

Various Primary lens luxation ADAMTS17 Mid-Late

X (Farias et al. 2010)

Beagle Primary open angle glaucoma ADAMTS10 Early X (Kuchtey et al. 2013)Russel Terriers Spinocerebellar Ataxia KCNJ10 Early X (Gilliam et al. 2015)Various von Willebrand’s Disease Type I vWF Early Xα (Brooks et al. 2001)Shetland Sheepdog von Willebrand’s Disease Type III vWF Early X (Rieger et al. 1998)Various Gallbladder mucoceles ABCB4 Early Xα (Mealey et al. 2010)Samoyed Dog Hereditary Nephritis/Glomerulopathy COL4A5 Early Xα (Zheng et al. 1994)

*AR – Autosomal Recessive Mode of Inheritance*AD – Autosomal Dominant Mode of Inheritance*XL – X-linked Mode of Inheritance β Patented testα Displays incomplete penetrance in carrier individuals

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5.3 Polygenic disorders and disorders with unknown mode of inheritance

Disorders for which the mode of inheritance is polygenic (two or more genes are involved in inheritance) are more difficult for breeders to manage. In polygenic disorders several genes must combine to cross a threshold producing an affected individual. If phenotypically normal parents produce affected offspring, both should be considered to carry a genetic load that combined to cause the disorder. Hip dysplasia, elbow dysplasia and brachycephalic obstructive airway syndrome are classical examples of polygenic disorders. To complicate matters, polygenic disorders are frequently complicated by environmental factors that may play a significant role in the expression of the disorder. In the case of hip dysplasia, environmental factors such as over-nutrition may have larger influences than genetic factors.

There are no known tests for polygenic disorders in dogs or cats and this would be very difficult to achieve in future. Some attempts at genetic testing (the so called genomic based test) for hip dysplasia in German Shepherds Dogs failed to assess individual risk for canine hip dysplasia (Manz et al. 2017). The best tool we have regarding such defects is to discriminate against affected individuals. Breeding phenotypically normal individuals with other phenotypically normal individuals of which preferably all or most littermates are also phenotypically normal, has the greatest chance of effective selection against the numerous defective genes of the disorder. Access to the family tree and pedigrees may assist in assessing risk and selecting phenotypically normal animals. This process is more easily performed if an open health database is available and to a lesser extent if only a semi-open health register is available. In cases where there is no open or semi-open health registry then breeders have limited options. In this case breeders must rely on their own results combined with the few they are willing to work with them and disclose their results also. Although not ideal, this is at least a start and better than having no results at all with no options.

6 COMPLEXITY OF INHERITANCE AND VARIATIONS TO THE MODE OF

INHERITANCE

In addition to environmental factors, there are other factors that may influence expression and severity of a genetic disorder. These factors may be present in various modes of inheritance and complicates the identification of carriers in breeding stock populations.

a) Variable expression

As the word implies, some defects may have variable expression with reference to the nature and severity of the phenotypic expression of the disease e.g. short tail in corgis thus same litter same parent’s different phenotypic expression in puppies. Multifocal retinopathy 1 is an autosomal recessive disease prevalent in various breeds such as Boerboel, Shepherds and Mastiffs (Guziewicz et al. 2011; Hoffmann et al. 2012; Gornik et al. 2014; Donner et al. 2016). There is enormous variability between breeds and individuals of the same litter with respect to precipitation of symptoms, severity of the symptoms and the rate of progression in affected individuals. While the symptoms and severity vary in affected individuals, carrier or clear individuals will not present any symptoms.

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b) Incomplete penetrance

Penetrance refers to the phenotypic expression of a disease. Incomplete penetrance refers to absence of disease despite presence of the dominant disease gene. For example, a dominant disease gene that causes disease 50% of the time is 50% penetrant. Thus, absence of a disease in a dog's parents and grandparents does not indicate absence of an incompletely penetrant dominant disease gene; however, even an incompletely penetrant dominant disease gene should have caused the disease to surface somewhere in the animal's ancestry. This phenomenon complicates pedigree interpretation when working with dominant diseases, because phenotypically “normal” animals may not be genotypically normal (carriers of the disease) and there is no way of knowing unless the symptoms become apparent or the individual has been genotyped. This condition has been associated with the concept of a defect skipping generations. Von Willebrand’s disease is a good example, where dogs affected by the disease present bleeding symptoms, however carriers present a variety of severity from excessive bleeding to none at all (Christopherson et al. 2014). Another good example of incomplete penetrance is gallbladder mucoceles/hepatobiliary disease in Shetland Sheepdogs (Mealey et al. 2010), where there is no clear link between the development and non-development of cholecystitis in carriers, while all affected individuals present the disease and share the progression timeline.

7 DIFFICULTIES IN RIDDING A POPULATION FROM GENETIC DISORDERS

To the uninformed, the notion exists that simple selection against a hereditary defect or disorder will ultimately rid the population of such defect and that failing to achieve confirms that the breeder is not selecting sufficiently against the disorder at all or breeding with affected animals. This misconception needs to be adequately addressed. In the absence of a genetic test that can identify carriers it is very difficult to rid populations from deleterious genes (Farrell et al. 2015).

To address this, we use the example elegantly explained by Gubbels (2014), where the mode of inheritance is autosomal recessive and the case is heritable cataracts. Assuming that 4% of the population is confirmed by phenotypic testing to suffer from heritable cataracts, using Hardy-Weinberg Equilibrium theorem (Stark 2005), it is calculated that 32% of the population will be carriers. The problem however is that we do not know who those carriers are because carriers and non-carriers are equally unaffected, thus we select our breeding stock from an unknown population. Assuming this cataract affected population is subject to strict selection against by means of phenotypic testing for ten generations, each generation being 2 years, after 20 years the prevalence of cataracts will statistically be reduced to 0.5% but the number of carriers will still be as high as 12.5%, and even after 100 years of selective breeding the population will still be composed of 4% carriers. The example assumes random mattings with healthy individuals only - in the real world this does not happen.

Breeders, understandably, select a specific stud that display outstanding breed-typical qualities that meets all the required health requirements as stipulated for the breed including in this case having passed the phenotypic cataract test. If 32% of the population are carriers, there is a good chance that a third of all the “superior studs” may be carriers. It is not inconceivable that this breeder may decide that their chosen superior stud should sire at least 10% of the litters in the next generation. The effect of this is that in one generation the

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number of affected animals may increase vastly (Gubbels 2014). The opposite is of course also true. It may have been that the breeder was lucky, and his selected superior stud was not a carrier and then of course this lucky breeder will have made more progress than in our random mating example. The problem lies in the law of averages and inability to identify carriers, only 2/3 breeders will have this luck. However, there is another reality that manifests itself in many hereditary disorders.

The following example sketches the outcome of a very unlucky breeder. Assuming this specific hereditary cataract manifests itself at an advanced age, then it may be that the breeder’s superior stud may indeed have passed five annual phenotypic tests and is identified as an affected animal at the sixth test. Using simple Mendelian laws of inheritance, this means that instead of having produced an estimated average of 25% of carriers in the unlucky breeders’ example above, this very unlucky breeder has now bred for several years with a stud resulting in all his offspring being carriers and depending on the dam being a carrier or not, some being affected. Needless to say, this population of this breeder is now set back many years in a single generation with many unhappy customers and unfortunate pets in tow. Remember also that this breeder was very unlucky because he had an initial chance of only 4/100 to select an affected superior stud. A real-life study involving the result of selective breeding using a screening programme on the prevalence of congenital hereditary sensorineural deafness in the Australian Cattle Dog confirms the hypothetical examples above. In this study, despite 10 years of testing, no substantial reduction in prevalence of congenital hereditary sensorineural deafness was evident in 608 pups from 122 litters from 10 breeding kennels (Sommerlad et al. 2014). While phenotypic testing remains important, it is clear that bad luck may visit any breeder with the best intentions. More importantly, while phenotypic selective breeding against hereditary disorders has its limitations, the lack of selection against disorders has a greater devastating effect overall. It also shows that concerted breed strategies are required to combat genetically based disorders.

8 BREEDING RECOMMENDATIONS BASED ON MODE OF INHERITANCE

8.1 Breeding recommendations for dogs that are potential carriers of autosomal

recessive heritable disorders for which there is no test available

Siblings, parents and closely related individuals of dogs with known recessive disorders may be potential carriers. If there are no genetic tests available to identify carriers for autosomal recessive disorders, the only thing that can be done is to test for the phenotype and having identified affected individuals, obtain knowledge of pedigree backgrounds. An open health database is the best method for objectively disseminating this information. Based on the outcome of the relative risk pedigree analyses, the animals should be categorised as high or low risk regarding carrier status (Collins et al. 2011). Unfortunately, such risk evaluation can only be assessed using huge databases of accurate health improvement schemes for a specific disorder in quest. Furthermore, such risk evaluation is not perfect. Ideally, superior quality animals (animals that performed well according to breed surveys) of low risk should be bred to other quality animals of low risk. If this is not possible, superior animals of high risk animals, should be bred to lower risk animals. Animals of high risk should not be bred to each other. Ideally, average animals of high risk should preferably not be bred as the long-

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term aim is to ultimately only breed low risk to low risk animals. This practice will diminish the frequency of the defective allele in the population. However, total eradication of genetic defects without being able to conclusively test for carriers proves difficult to impossible. Vastly reducing the number of carriers and affected animals is a more realistic expectation. Lastly, any system of dog breeding that sets priorities for health and welfare must, almost by definition, be flexible. It must review its selection criteria, including breed standards, in light of the latest disease prevalence data (Collins et al. 2011).

8.2 Breeding recommendations for dogs that are potential carriers of autosomal

recessive heritable disorders for which there is a test available

The control of genetic disorders for which there is a test is simple. In this instance the purist approach of only breeding non-carriers is not ideal. This is because there exists a tool to both maintain genetic diversity and preserve the genes of quality individuals without the risk of producing affected animals. When there is a test available for a specific recessive genetic disorder, mating of quality carriers to non-carriers is permissible. In exceptional cases it may even be acceptable to mate affected with non-carrier in order to preserve genetic diversity without risk of producing more affected animals. With a recessive disorder this is permissible because there is zero risk of producing affected offspring but there is a real risk of producing more carriers. Therefore, this strategy of using known carriers in a breeding program should be considered a temporary measure to retain genetic diversity in the short term with the long-term goal of not breeding with carriers and eliminating the defective alleles from their breeding stock.

8.3 Breeding recommendation for dogs with Autosomal dominant and X-linked

dominant heritable disorders

Do not breed

8.4 Breeding recommendations for dogs that are potential carriers of Autosomal

dominant and X-linked dominant disorders for which there is no test available

Control is same as for autosomal recessive heritable disorders

8.5 Breeding recommendations for dogs that are potential carriers of Autosomal

dominant and X-linked dominant disorders for which there is a test available

Control is same as for recessive autosomal heritable disorders

8.6 Breeding recommendation for dogs with sex-linked recessive heritable disorders

Do not breed.

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8.7 Breeding recommendations for dogs that are potential carriers of sex-linked






8.9 Breeding recommendation for dogs with sex-linked recessive heritable disorders

Do not breed.







9 NEED FOR BREED SPECIFIC AND DISEASE SPECIFIC COUNSELLING

The complexity of inheritance and variations to the mode of inheritance may require breed specific, disease specific approaches and the implications of testing considered on a breed by breed and disorder by disorder basis. Veterinarians, breeders and breed authority officials may not have the required knowledge on how to interpret the test results and the implications it has, it is therefore advised that this effort is a collaborative one involving breed specialists and canine geneticist following scrutiny of the literature and collaborative efforts with specialists abroad. Genetic testing laboratories that report results also have the responsibility to provide sufficient descriptive text in their reports aiding all parties in correctly interpreting results on a disorder by disorder basis.

To illustrate the difficulties in interpretation of results the example of degenerative myelopathy (DM) is used. The mode of inheritance strongly suspected to be autosomal recessive. The test detects the presence or absence of the mutation associated with DM within the gene SOD1, for which the alleles c.118A appears to be widely distributed in the overall canine population. In reality, studies have shown that a small percentage of dogs with confirmed DM may indeed be heterozygous for the SOD1: c.118A allele. Also, although almost all confirmed DM affected dogs will be SOD1: c.118A homozygotes, not all homozygotes will ultimately develop confirmed DM in old age. The problem now emerges as to how to use the DM test in a breed and what is the fate of puppies that test positive for DM.

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Owners would be reluctant to buy a known homozygote for DM and the breeder would be reluctant to breed with them and it also unsure whether the homozygote will indeed ever develop clinical DM. Routine testing in this case would then lead to the destruction of a large number of puppies which would either develop the condition at an advanced age or perhaps never. This risk and uncertainty prompts many breeders not to test for a disorder such as DM as it has to many uncertainties and negative ramifications. Despite this all, it is however still concluded in populations where the SOD1: c.118A allele is widespread, that breeding to avoid the production of SOD1: c.118A homozygotes is a rational strategy (Zeng et al. 2014). Local population screens should be strongly considered in planning breeding strategies for this and many other defects. As a note of caution, reference to confirmed DM is required in this context as the clinical signs of DM may be indistinguishable from the cauda equina syndrome which also has a late onset. There is no genetic test for cauda equina syndrome and specialised diagnostic techniques are required for its diagnosis. Lastly to further complicate matters, the Bernese Mountain Dog has another allele (SOD1: c.118T) that is implicated in DM and some German Shepherd Dogs may have yet another allele. Similar complexities are present for other genetic disorders and all these require expert evaluation in the breed in order to make sensible breeding recommendations.

Interpretation of literature regarding heritable disease and mode of transmission may both be complex and misleading for lay persons. For instance, an article published on epilepsy in Irish Wolfhounds suggested that assuming all affected dogs have the same form of epilepsy, the simplest description of the complex pattern of inheritance observed is autosomal recessive, with incomplete penetrance and male dogs at increased risk. (Casal et al. 2006). This article should only act as catalyst for genetic test development and surveillance of the local dog population for the epilepsy problem. Following that, a sensible breed recommendation can be made.

10 LIMITATIONS OF DNA DISEASE TESTS

Performing genetic tests should be done in such a way that it attempts to preserve genetic diversity whilst stopping the spread of the genetic disorder. By merely eliminating the defective allele without maintaining genetic diversity one may correct one problem but inadvertently create new ones.

Caution needs to be exercised when implementing new tests based on preliminary or assumed relationship between a disorder and linked marker tests. The example of goniodysgenesis and its relationship to the development of glaucoma in the Border Collie is given. Although goniodysgenesis is strongly associated with an increased risk of developing primary closed angle glaucoma, some Border Collies with goniodysgenesis never go on to develop glaucoma. In humans, only 10% of people diagnosed with anatomically closed angles will develop glaucoma (Wang et al. 2002). Until the relationship between goniodysgenesis and glaucoma is clarified, basing breeding strategies, solely on the presence of goniodysgenesis or on a specific DNA test for goniodysgenesis, may not reduce or eliminate glaucoma from the Border Collie population, especially if glaucoma is genetically heterogeneous and/or epistatic involving several different loci in this breed (Farrell et al. 2015). This does not however mean that the test should altogether be abandoned. Breed specialist groups should consider placing such test under investigation. This can be done by testing populations over a

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period of time to establish causative link to disease (expressed in percentages) and base their recommendation on the subsequent finding before making a blanket decision.

Genetic heterogeneity within and between breeds may also influence the utility of DNA testing. This is because tests may be breed-specific or even sub-population specific as exemplified by testing for progressive retinal atrophy (PRA), a collective of genetic eye disorders that share similar symptoms (Farrell et al. 2015). There are 33 causative mutations associated with PRA related diseases (OMIA) and there is increasing evidence that multiple different forms of the disease segregate in more than one affected breed, and possibly within an affected individual (Miyadera et al. 2012; Downs et al. 2014a). This means that two or more distinct mutations may be present in a single breed and an individual may possibly possess a mutation not targeted by the specific DNA test for which it is recommended. As more screening projects are performed, this challenge can be mitigated by performing all available DNA tests for the various forms of PRA to assess all clinical cases, regardless of the recommended breed specific test (Downs et al. 2014a). As technology improves and more mutations are identified, it will become more cost effective to screen individuals for all the characterised mutations available. However, we should be reminded that there are potentially more diseases that have not been characterised, and some of these could be PRA related. Genetic heterogeneity may also influence test results for dilated cardiomyopathy in Doberman Pinschers and other breeds (Meurs et al. 2008).

11 ESTIMATED BREEDING VALUES AND GENOMIC SELECTION

The estimated breeding values (EBV) measures the potential of an animal to pass a specific trait to its offspring using the dog’s results on phenotypic tests and or the results of relatives in conjunction with their pedigree relationships. This method is useful for selection on complex disorders influenced by multiple genes and environmental factors as it gives an estimate of the likelihood of development of a disorder. The premise is that selection based on EBVs rather than on individual phenotypes is more likely to increase rate of genetic progress in complex disorders where EBV is used as selection criterion (Lewis et al. 2013; Wilson et al. 2013). The EBVs for hip and elbow dysplasia are widely used in some countries. Reliability of the EBV relies on the accuracy of the data, size of data base as well as accuracy of parentage. Pedigree based information can therefore be replaced by using relationships based on genome-wide markers and the EBV calculated from this genomic selection is then termed genomic EBV. Because the vast majority of puppies that are bred by breeders are destined for the pet trade and only a small percentage (usually around 10% or less) will be screened for genetic disorders as potential future breeding dogs. This small number makes it difficult to obtain meaningful EBVs.

12 ADDRESSING THE ISSUE OF INBREEDING

It is important that veterinarians have a good understanding of the concept of inbreeding. This will help them in appropriately responding to claims of inbreeding and bad breeding and so forth. Inbreeding means the mating of individuals which are more closely related to each other than is the case for the average of the population from which they come. Inbreeding is not just a man-made phenomenon but occurs naturally in many species as well. Wolves and

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various wild dogs may become intensely inbred because of isolation from other populations, overhunting or the all too common destruction of habitat and extermination of their prey.

All dog breeds exhibit an extent of inbreeding, as inbreeding is achieved when individuals begin to phenotypically resemble one another as an increase in homozygosity is reached. This is how dog breeds were developed in the first place. All dog breeds have to some extent been inbred, the extent of which depends on location and population size (Calboli et al. 2008). From this perspective, breeders have suggested that some extent of inbreeding is required to accomplish desired breed standards. It encourages breeders to make use of outstanding studs and bitches, to genetically entrench (fix) the good characteristics of the parents in their offspring. A level of homozygosity is achieved, by the uniformity observed in the offspring in addition to the high degree of resemblance of offspring to both parents. This breeding strategy is termed line breeding.

When line breeding is practiced and there is a high degree of phenotypic resemblance between the offspring and ancestors, a “bloodline” has been established. Line bred dogs are more likely to breed true. This means that they produce offspring which possess many of the good qualities they have themselves. Prepotency is sometimes used by breeders as breeder “slang”, synonymous to breeding true or having the ability to produce offspring bearing a strong resemblance to itself. The term prepotency is mostly used for studs. Achieving homozygosity using judicious inbreeding is therefore a must in the quest for progress towards improving the breed standard.

While the merits of inbreeding have been adequately discussed, it is prudent that the risks are discussed too. The extent (intensity) of inbreeding is a subject of much debate because incessant inbreeding may reveal detrimental effects as recessive alleles accumulate, and traits become fixed within a breed. Inbreeding can also limit the genetic pool when popular studs are overrepresented (the so-called popular sire syndrome) in stud registers and less popular studs used sparsely or not at all. For this reason, it makes sense that breeding authorities should impose limitations on the number of offspring per stud, thus reducing the popular sire effect and promoting increased genetic variability on a population-wide scale. Such restrictions on sires are already in place for the German Shepherd Dog (Guyader 1989).

Lack of genetic diversity may have serious consequences (Leroy 2011). It may decrease the statistical chance of finding individuals within the inbred population that retain the genes to resist disease if new diseases emerge. A common consequence of intense inbreeding is decreased reproductive success characterised by low conception rates and increased mortality rates in the offspring.

What recommendation is most appropriate regarding inbreeding?

We simply refer the breeder to follow the breeding recommendations stipulated by their respective breed authorities. Inbreeding is measured using coefficient of inbreeding (inbreeding coefficient) also called Wright’s coefficient. It may be expressed either as a percentage or as a proportion and is usually denoted by the term F. Many breed authorities have available calculators from which the inbreeding coefficient can be calculated prospectively by entering the kinship of the prospective breeding pair. These breeding authorities then also give recommended inbreeding coefficients which should not be exceeded.

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Diversity coefficients (DC) are determined between individuals based on their genotypic profiles. There are different diversity coefficient standards/limits from which breeders can gauge which mating pairs are most ideal for each breed based on the population genotypic diversity. These tools could be useful when deciding between studs. The measurement of diversity coefficient will provide you with a value dependent on the number of markers employed and the genetic diversity of the breed population. If the latter is unknown, and your gene pool is limited, it is suggested that the individual with the lower DC value be used, as this indicates that there is greater genetic discrimination between the proposed mates.

13 MOST FREQUENTLY ASKED QUESTION BY PET OWNERS

13.1 Is this disorder a genetic disorder?

This question is frequently asked by pet owners that are disgruntled upon discovering from their veterinarian that their pet dog has some or other “condition” or ailment. The intent of the pet owner may be to confront the breeder with proof of a heritable condition to seek replacement or compensation for the purchase price and veterinary expenses allegedly caused by the diagnosed condition. They may seek written confirmation from their veterinarian to put their findings in writing and insist that reference to “genetic in origin” features in the document. Some pet owners are also willing to take legal action. First and foremost, it is important to ask the pet owner if there was a purchase sales agreement between themselves and the breeder. To avoid these disputes all owners and breeders should be encouraged to have in place properly detailed purchase sale agreements which clearly state terms and conditions of sale and should include eventuality of genetic disorders and latent defects. The dispute can then be easily resolved as the terms of the agreement are legally enforceable. In many cases breeders are willing to replace or partially compensate beyond the terms stipulated when approached. In the absence of a binding contract, assumption of a “voetstoots” transaction is usually made. Proper contracts protect both the interests of the breeder and pet owner. It is not always possible for a competent breeder to guarantee against all possible genetic disorders. They should therefore stipulate what guarantees they can provide. Explanation by veterinarians of the difficulties regarding control of genetic disorders to their owners often immediately defuses the situation as they then understand that there probably was no malicious intent or negligence on behalf of the breeder. It is imperative that a veterinarian be very careful when using the word “genetic” unless they are willing to accept all its consequences. A condition such as blindness may be genetic, however could be developmental, caused by injury or disease. Accurate diagnosis is always required. Furthermore, the term “genetic” is interpreted by many pet owners as perfectly preventable. This misconception should also be addressed by veterinarians as proper communication by an informed veterinarian goes a long way in resolving these issues and preventing a multitude of unpleasantries.

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14 MOST FREQUENTLY ASKED QUESTIONS BY BREEDERS

14.1 Can I breed with the affected dog?

Irrespective of mode of inheritance, it is ill advised to recommend breeding with affected individuals as it may lead to the increased frequency of carrier or affected individuals in the population. Theoretically if the mode of inheritance is autosomal recessive and a test is available, the affected animal could be bred to known non-carriers, as all offspring of this pair would be carriers. Such a breeding strategy could only be justified in dogs of exceptional quality where only a very small genetic pool is available. Should this breeding strategy be implemented, the offspring of these pairs should be tested for a number of generations to ensure that the carrier population is reduced over time. Astute geneticists will vehemently defend this concept as they have the overall genomic health of the breed in mind (Gubbels 2014). Furthermore, failing to accept this has led to increasing frequency of one or more deleterious alleles in the population.

14.2 Does the disorder come from the male or the female in the combination?

Depending on the mode of inheritance of the disorder it may be from either or from both and their contribution may be equal or not. If the mode of inheritance is known and there is a test available for the gene, then this question can be answered conclusively. If there is no test available and the mode of inheritance is not known, it is very likely that the disorder is a recessive disorder. It is safest to assume that both parents are likely carriers if the genetic status of your stock is unknown. Owners of either the male or female may often claim that they have never seen this defect in the males’ or females’ line and therefore, in their opinion, is extremely unlikely that their dog is the carrier. Whilst this observation of the breeder might be true, it is still sound to assume that both parents are likely carriers unless conclusively proven otherwise through genetic testing. If the condition is X linked and the offspring is male, it is likely the bitch that carries the mutant allele.

14.3 Can I use the parent combination of an affected dog?

The answer to this question depends on whether the mode of inheritance is known and whether there is a test available for the disorder. It would be advisable to test the pair as it is not recommended to breed carrier to carrier, carrier to affected or affected to affected. It is not possible to estimate the parent combination in an autosomal dominant condition without genetic testing if previous mating’s have not been performed.

14.4 Can I breed with the parents again if either or both are known or suspected carriers

of a disorder?

The answer to this is yes and no, depending on whether the mode of inheritance is known and whether there is a test available to identify carriers. See section on mode of inheritance and breeding recommendations.

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14.5 Can I breed with siblings of the same parent’s pair of affected individuals?

The answer to this is yes or no, depending on your breeding strategy, whether the mode of inheritance is known and whether there is a test available to identify carriers.

14.6 Are there conditions not documented that may potentially have a genetic origin?

Any condition may have a genetic base to it. Susceptibility to disease may have its genetic origins. Conditions which are over-represented in certain breeds are likely candidates for genetically linked disorders. Besides selecting against the phenotypical display of the suspect disorder, there is nothing else one can do. Health registries usually do not exist for conditions suspected to be of genetic origin unless they have become very prevalent in the breed.

14.7 Assuming you test a litter before sale for a whole panel of genetic disorders what do

you recommend be done with either proven carriers or affected individuals?

Depending on the disorder and its mode of inheritance, you will do; nothing, sterilize the individual involved, euthanize the individual or let it go to the new owner (or keep the individual) provided all parties have been fully informed of the possible consequences of keeping affected individuals.

14.8 What possible explanations are there if a test result just does not make any sense

according to its mode of inheritance?

The example used is as a puppy testing as a carrier/affected for a genetic disorder despite both the parents being clear for the condition. There are many variables that can affect the accuracy of a test result; chain of custody is most often the cause through human error by the veterinarian, breeder or laboratory (although reputable laboratories implement quality control points to prevent this possibility), type of sample taken (whole blood not contaminated through a transfusion is the best sample type), and contamination of sample (if you have sampled from a common area that is contaminated by other canine/human DNA) are all possible. It is recommended that the chain of custody for the sample submitted be followed closely to identify whether this is the introduction of the variable. Due to the unlikely event that the mutation detected is caused by a spontaneous mutation (a rate of 0.1-100 per genome per sexual generation) (Drake et al. 1998), we recommend testing parentage first. Incorrect parentage allocation is more common thang generally thought, especially if multiple studs are kept in the same area. If the chain of custody and parentage checks out, it is advised to have the dam, sire and puppy retested. An error in the accuracy of a test is possible but will be dependent on the type of test performed, and the quality controls implemented or not implemented. Direct DNA tests are the most accurate laboratory test with accuracy above 99.9%, while secondary detection tests (which make the test more affordable, also known as linked tests, markers or indirect tests) generally exhibit an accuracy of >95% (false positives and false negatives are therefore possible albeit highly unlikely). Poor understanding of the variations in mode of inheritance such as incomplete penetrance or multimodal (more than one allele involvement) modes of inheritance may complicate test result interpretation;

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degenerative myelopathy is such an example in different breeds (Zeng et al. 2014). Theoretically, natural chimaerism is possible in the dog but admixture of blood (via blood transfusion) leading to haemopoeitic chimaerism is a common and more realistic cause of chimerism and potential erroneous results.

14.9 Is it possible for parents that tested negative (using a direct test) for a specific

disorder, to produce a puppy testing positive for that specific genetic disorder?

Given; no mistakes were made regarding chain of custody, parentage, identification and laboratory error and assuming the test is 100%, theoretically this is possible for autosomal dominant genetic disorders but are extremely rare, through the formation of a new mutation. This mutation would develop in the maternal or paternal gamete during meiosis at a rate of 0.1-100 per genome per sexual generation (Drake et al. 1998). The statistical probability that the same mutation in either of the parent gametes might repeat itself is even more unlikely and so one could assume that the parents are safe to use again.

14.10 Should the breeder continue spending money on tests if all his dogs in their kennel

are proven clear for a particular genetic disorder?

If all the tests are 100 % accurate, the obvious answer is that no further testing is required. Some pet owners may however insist on a clear test before purchase necessitating continued testing of offspring emanating from clear parents. However, when using outside studs or introducing new stock it is safer to only trust the clear status based on a clear test and not relying on a clear status based on the parents either allegedly being clear or confirmed being clear. This is because there may be error in the parentage of the individual involved or some other mistake. Overseeing this simple measure may lead to the reintroduction of a genetic disorder in their lines with the breeder only realising the mistake some generations later.

14.11 Is total eradication of genetic defects possible?

Only single gene genetic defects for which there are tests available can be totally eradicated. For other defects for which there is no test available yet, only reducing the prevalence of genetic disorders is a realistic expectation.

14.12 Are there potential risks involved in eliminating defective genes from a genetic

pool?

Using genetic testing as a tool to remove individuals from a population is a risk as most dog and cat breeds have a closed stud book with a limited gene pool and will lose further genetic diversity if you remove individuals completely from the population to weed out the undesirable traits. Another potential adverse effect is where there appears to be a correlation between absence of one disorder to the presence of another. This may be either purely incidental or genetically linked.

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14.13 Why does it happen that an entire litter proves to be carriers or affected whilst only

25% of the litter were expected to be carriers or affected?

When dealing with autosomal recessive disorders the expected outcome of breeding a carrier to a non-carrier is that 25% of the offspring may be carriers and likewise when affected individuals are bred to a carrier then 25% of the offspring are expected to be affected. This is a common misconception by particularly breeders and sometimes even veterinarians leading to incorrect speculation about other possible causes having played a role other than pure statistical chance. This phenomenon is better explained because the 25% probability is the probability per fertilisation and refers to an average over a large number of fertilizations. It is important that veterinarians recognise this misconception and misinterpretation of statistical outcome and explain it to the breeder. This is because there are many instances where a breeder notices all puppies within a litter to be affected by a defect whilst they were only expecting 25% affected puppies. Upon the finding of yet another litter with a disproportionately high representation of the defect they are finally convinced that there must be another explanation. They then remember that their veterinarian had advocated a new more effective systemic anti parasiticide (or other medication) and the breeder starts to suspect that this may be the cause. Following some social media messages about other breeders having experienced the same, their suspicion is then finally “confirmed” and a social media campaign regarding unfounded claims of teratogenicity is likely to follow.

Another example of statistical chance which may confuse breeders is the finding of skewing of the sex ratio within a litter. The breeder may have two consecutive litters where all the sexes of the puppies in an eight and 13-puppy litter may be male. To the breeder this statistical mishap to have been purely incidental (statistical) seems too great and they start suspecting other mysterious forces at play.

14.14 Are all tests developed for a defect in one breed applicable to another population

where there is little admixture between the two populations. For instance, a test

developed for Golden Retrievers in the USA being applicable to the European

Golden Retriever population?

Theoretically all mutations could be identified in all breeds as all breeds originated from a common ancestor. However, mutations have accumulated and become fixed in certain breeds due to the breeding strategies implemented over thousands of years. While some genetic diseases have been restricted to one breed, like dry-eye-curly-coat-syndrome in the Cavalier King Charles Spaniel, other diseases seem to be common amongst breeds, like progressive retinal atrophy (PRA). While some forms of PRA, are breed specific, many other forms have been identified in various breeds and there is increasing evidence to suggest that the different forms of PRA have segregated together (Miyadera et al. 2012; Downs et al. 2014a; Farrell et al. 2015). This suggests that many more distinct mutations may be present in a breed and these mutations may not be restricted to this one breed. While the prognosis sounds depressing, this may be good news for breeders. As technologies evolve and become more affordable, soon, breeders will be able to test multiple well characterised genetic diseases. Full genome analysis may become an affordable reality to predict future genetic disorders.

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15 CONCLUSIONS

There is an increased public awareness of genetic disorders in dogs and cats and therefor the importance of testing for genetic disease and participation in health improvement programmes needs to be encouraged. Breeders of pedigree dog and cat breeds face serious challenges in combating inherited disease. Small animal practitioners should recognise these challenges and explain this to pet owners. They should familiarise themselves with the more common inherited disorders and the tools available to improve genetic health of dog and cat breeds. Inherited disorders are unfortunately an inconvenient reality in both man and animals. Veterinarians that have a specific breed interest should avail themselves in collaborative efforts with breed interest groups to create breeding strategies with the aim of significantly reducing inherited disorders.

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