A novel possibility of molecular diagnosis for the English Cocker Spaniel

by Dr. Bernd Hackauf

Introduction

More than 1000 inherited diseases have been described in dogs^[1,2] representing the major challenge of breeding purebreed dogs in the 21^st century. In a recently published review on inherited defects in pedigree dogs, which was primarily addressed to  disorders related to breed standards, it has been reported, that each of the top 50 UK Kennel Club registered breeds was found to have at least one aspect of its conformation predisposing it to a disorder; and 84 disorders were either directly or indirectly associated with conformation^[3]. Molecular selection methods currently allow an efficient genetic management, however, only for a minor part of the known genetic defects in dogs.

A novel molecular assay has most recently been established addressing an important category of kidney diseases in dogs, Juvenile Nephropathy alias Juvenile Renal Dysplasia. Here, the state of the art of science and technology concerning the genetic management of this kidney disease by means of a molecular genetic approach, which provides an invaluable contribution to the combat against inherited diseases in dogs, will be reported with particular emphasis on it's application in the English Cocker Spaniel.

Two kidney diseases of the dog

The primary function of the kidney is to filtrate the blood with respect to a variety of waste products produced by metabolism and to excrete wastes such as urea and ammonium. In healthy kidney's many of the kidney's functions are accomplished by relatively simple mechanisms of filtration, reabsorption, and secretion, which take place in the nephron, the functional unit of the kidney. The nephron's initial filtering component is the renal corpuscle, which is composed of a glomerulus and Bowman's capsule

Nephropathy refers to damage to or disease of the kidney. The Familial Nephropathy (FN) is a juvenile-onset fatal kidney disease, which is known in English Cocker Spaniels for more than 50 years^[4]. FN in the English Cocker Spaniel is caused by a type IV collagen defect within the glomeruli. The mutation causing this disease has been identified, and use of a DNA test for the mutation permits eradication of FN in the English Cocker Spaniel. A defined selection scheme of breeding animals allows to supplement the breeding strategies in order to avoid a breeding and selling of puppies affected by FN.

Juvenile Renal Dysplasia (JRD) is another important category of kidney diseases in canines and is sometimes referred to as Juvenile Nephropathy. Dysplasia is defined as abnormal growth of cells and organs. In the case of JRD the kidney fails to develop properly during embyogenesis in the womb^[5]. At birth immature structures consisting of undifferntiated fetal cells or tissue types are found in the kidney^[5].

JRD has been reported from many breeds. Breeds most notably affected with JRD include Shih Tzus, Lhasa Apsos, Soft Coated Wheaten Terrier and Miniature Schnauzers^[5]. Standard Poodles and the German Shepherd Dog are affected as well. JRD has previously been detected in the Cocker Spaniel^[6]. Sweden leads a controversial discussion on Progressive Nephropathy (PNP) due to Renal Dysplasia in English Cocker Spaniels.

JRD in affected breeds share a common phenotype, which is characterized by immature glomeruli, and/or tubules and persistent mesenchyme, and defects in the renal cortex^[5]. Up to now a wedge biopsy revealing dyspastic lesions including abnormal ducts and glomeruli allowed a definitve diagnosis of JRD^[5]. Individuals with an abnormal biopsy can, however, be asymptotic, showing no signs of the disease. On the other hand, affected animals may present with classic signs of chronic end stage renal failure or somewhere in between these two extremes^[7].

Due to this wide range of symptoms and pathological findings of this disease, the mode of inheritance is widely discussed. A recessive mode of inheritance has been prosposed e.g. for the Shih Tzu^[8], a genetic model which is mentioned for the Bernese Mountain Dog as well. In contrast, a more recent study suggests a dominant inheritance with incomplete penetrance^[7][9].

Development of a DNA-Tests for JRD

The dog genome encodes for more than 19.000 genes^[1]. One approach to extract from this tremendous amount of information excatly the particular gene sequence associated with the expression of a trait like a certain disease is to analyze candidate genes. A gene becomes a candidate in research, if there is knowledge e.g. from model organisms on the association of the gene with the expression of a trait like a certain disease.

This approach has now successfully been applied by the geneticist Dr. Mary Whiteley to detect the gene governing JRD in dogs^[9]. Most notably and beneficial for Dr. Whiteley's research was, that the mouse model displays a JRD phenotype identical with that observed in dogs. Sequence analysis of initially 6 candidate genes allowed Dr. Whiteley finally to identify the JRD associated mutation in the gene Cyclooxygenase-2 (COX-2) of Lhasa Apsos and Shih Tzus, respectively. Furthermore, genetic variants (alleles) of the COX-2 gene have been identified in other breeds like the English Cocker Spaniel, which are affected by JRD as well^[9]. In contrast, breeds which have yet not been reported to be affected by JRD revealed only the non-mutated wild type allele of COX-2. Based on these findings, Dr. Whiteley has established a DNA assay which allows to discriminate the different COX-2 alleles. This assay enables to genotype dogs at the COX-2 gene with high precision which, subsequently, allows to classify the genetic predisposition of a dog for JRD.

The COX-2 genotypes are scored analogous to prcd-PRA or FN , i.e. (A) clear: the dog carries no mutant COX-2 allele, (B) carrier: the dog carries one copy of a mutant COX-2 allele, (C) homozygous mutant genotype: the dog carries two copies of a mutant COX-2 allele. With results (B) and (C) the dog may be potentially affected by JRD and could pass the mutant allele on to its progeny^[5].

The results of this DNA assay should be carefully used for any breeding decisions, which largely depends on the frequency of mutant COX-2 alleles in a breeding population. In a North American Shih Tzu population, for instance, JRD occurs in high frequency. Among 74 randomly selected dogs only 16% revealed no histological symptoms of the disease^[7]. In populations like this a comprehensive selection of carriers would be the worthest breeding decision, as it would sustainably narrow the gene pool. Matings between dogs clear of any detrimental mutation in this and other genes should be a long term goal. However, this goal can be reached by using the available DNA assay's together with cool-headed breeding decisions, which should be addressed mainly to breed and sell puppies not affected by a genetic inherited disorder and not to endanger a breed by an artifical reduction of genetic diversity.

A case study of COX-2 genoytpes in English Cocker Spaniels

The published COX-2 genotype of Lochdene Time Keeper represents a first but responsible contribution of Monika Bollinger to evaluate the potential of the novel DNA assay for the English Cocker Spaniel. Current knowledge on COX-2 allows to deduce that mutations of this gene are inducing JRD in dogs. The described observations for two of Lochdene Time Keepers progenies fit to the reported association between JRD and a mutation in the COX-2 gene in dogs. Thus, the novel DNA assay appears to be an additional and valuable tool for breeding healthy English Cocker Spaniels. The publication of Monika Bollinger will hopefully encourage many if not all breeders of English Cocker Spaniels to use the genotyping service offered by DOGenes for their own dogs in order to reduce the risk for any puppy to suffer from JRD. Each additional COX-2 genoytpe of the English Cocker Spaniel population available to the public will increase our knowledge on the value of the assay for this breed.

This COX-2 assay provides a novel and precise opportunity to record a disease, which is phenotypic difficult to assess. For this reason, this DNA assay is particularly valuable for dog breeders. In the case reported by Monika Bollinger, the COX-2 assay delivered a possible explanation for the kidneys disfunction in two progenies descending from FN clear parents.

The low frequency of JRD affected progeny of this popular sire allows to expect, that the mutant variant of the COX-2 gene occurs in low frequency in the entire population of the English Cocker as well. However, Lochdene Time Keepers pedigree represents severeal famous sires and dams, which are well known in many other pedigrees of the breed. Thus, it appears to be wise to consider - as a preventive task - the comprehensive genotyping within the population of the English Cocker Spaniel to anticipate a major injury of the breed.

The risk of any newborn English Cocker Spaniel puppy to suffer from kidneys disfunction due to JRD can, however, already be minimized with the knowledge published by Monika Bollinger. Breeders should avoid inbreeding in general^[10] and particularly on Lochdene Time Keeper or any of his ancestors in order to reduce the probability of transferring the mutant COX-2 allele to a puppy. For the affected litter, the inbreeding coefficent based on the proposed pedigree of the parents and encompassing 10 generations is slightly higher than 8%, which appears to not dramatically high^[11], but higher than the average coefficient of inbreeding of F= 2,3% in a french sub-population of the English Cocker Spaniel^[12]. However, inbreeding applied for this particular litter has increased the probability to inherit the mutated COX-2 variant in one or even two copies to the progenies.

Summary

JRD is an important category of kidney diseases in canines. A DNA assay has recently been established to assess this inherited disease at the molecular level. This assay is applicable to many breeds including the English Cocker Spaniel. The novel DNA assay delivered a possible explanation for the kidneys disfunction in two English Cocker Spaniels descending from FN clear parents. This result demonstrates the potential of a comprehensive utilization of this molecular tool supporting breeding desicions in terms of healthy English Cocker Spaniels.

References

[1] Ostrander EA, Wayne RK (2005) The canine genome. Genome Res. 15:1706-1716

[2] Sargan DR (2004) IDID: inherited diseases in dogs: web-based information for canine inherited disease genetics. Mamm Genome. 15:503-506

[3] Asher L, Diesel G, Summers JF, McGreevy PD, Collins LM. (2009) Inherited defects in pedigree dogs. Part 1: disorders related to breed standards. Vet J. 182:402-411.

[4] Krook L. (1957) The Pathology of Renal Cortical Hypoplasia in the Dog. Nord. Vet.-Med. 9, 161-176.

[5] http://www.dogenes.com/jrd.html

[6] Felkai C, Vörös K, Vrabély T, Vetési F, Karsai F, Papp L. (1997) Ultrasonographic findings of renal dysplasia in cocker spaniels: eight cases. Acta Vet Hung. 45:397-408.

[7] Bovee KC (2003) Renal Dysplasia in Shih Tzu Dogs. Proc. 28^th World Congress of the World small Animal Veterninary Association.

[8] Hoppe A, Swenson L, Jönsson L, Hedhammar A (1990) Progressive nephropathy due to renal dysplasia in shih tzu dogs in Sweden: A clinical pathological and genetic study. J. Small Animal Pract.31:83 – 91

[9] Whiteley M. H. (2009) Compositions and methods for detecting Juvenile Renal Dysplasia or oxalate stones in dogs. Intl. Patent WO/2009/092171, filed January 22, 2009, and issued July 30, 2009.

[10] Hackauf B. (2009) Rassehundezucht auf dem Prüfstand. 1: Eine genealogische Bestandsaufnahme. Der Jagdspaniel 2/09:33-41

[11] Hackauf B. (2009) Rassehundezucht auf dem Prüfstand. 2: Eine molekulargenetische Bestandsaufnahme. Der Jagdspaniel 3/09:17-20

[12] Leroy G, Verrier E, Meriaux JC, Rognon X (2009) Genetic diversity of dog breeds: within-breed diversity comparing genealogical and molecular data. Anim Genet. 40:323-332.