S.L Debenham*, A. Millington†, J. Kijas+, L. Andersson+, M. Binns*
*Animal Health Trust, Lanwades Park, Kentford, Newmarket, Suffolk CB8 7UU, United Kingdom, †Genetic Sub-Committee, Irish Red & White Setter Club of Great Britain, and +Department of Animal Breeding and Genetics, Box 597, University of Agricultural Sciences, Uppsala, S-751 24 Sweden.
Seventy-six Irish Red and White Setter samples were tested for the recently published CD18 point mutation which manifests as CLAD in Irish Setters. Six carrier dogs were identified, all originating from a lineage within which sporadic deaths from symptoms consistent with CLAD had been observed. This demonstrates that the CLAD mutation is present in a significant minority of the Irish Red and White Setter population. Routine testing by breeders to identify carrier animals will enable the eradication of CLAD from the Irish Red and White Setter population.
Leukocyte Adhesion Deficiency (LAD) in humans is an autosomal recessive disorder, generally characterised by severe immune deficiency leading to infection and defective repair mechanisms after injury. Research has found that the clinical symptoms associated with LAD are caused by deficient expression of the b-2 integrin (CD18) molecule (Arnaout et al., 1984; El Habbal and Strobel, 1993), a glycoprotein involved in cell-cell adhesion events.
A bovine form of LAD (BLAD) was subsequently identified which, in affected cattle, displayed very similar symptoms to those in humans (Kehrli et al.,1990). Molecular studies proved that these symptoms were also caused by defective b-2 integrin expression, due to a missense mutation within the coding region of the gene.
Canine Leukocyte Adhesion Deficiency (CLAD) in Irish Setters was first identified in 1975 (Renshaw et al., 1975). The disorder again manifested as a severe immunodeficiency disease which in breeding experiments was found to display autosomal recessive inheritance, being fatal in affected dogs (Renshaw and Davis, 1979). Several animals displaying various forms of recurrent infectious and immunological complications, were found to have an aberrant expression of the CD18 molecule (Trowald-Wigh et al., 1992). The identification of the gene mutation responsible for the disease in Irish Setters was ascertained in mutational analysis of CD18 in Irish Setter CLAD pedigrees (Kijas et al., 1999). From this, a single missense mutation - Cys36Ser was identified which showed complete association with CLAD in Irish Setters (Kijas et al., 1999). The mutation is thought to be responsible for incomplete disulphide bonding within the b-integrin protein, causing defects in its function. CLAD is therefore genetically equivalent to both LAD and BLAD. This finding allowed the development of a DNA based diagnostic test for CLAD, which is commercially available and widely used by Irish Setter breeders.
The Irish Red and White Setter breed is derived from the Irish Red Setter working population. Originally in Ireland, only pure Irish Red Setters were used as show and working dogs. Generally, red and white puppies produced in Irish Red Setter litters were destroyed. In the 1970s, however, renewed interest in pure Irish Red and White Setters led to a revival of the breed. At this time there were less than 10 pure Irish Red and White Setters and it was from these few dogs that the present population is derived.
The first litters bred in the UK were in 1980 from 4 bitches imported from Ireland which were litter sisters. Three of these were returned to Ireland to be mated and the fourth was mated to an imported Irish Red and White Setter stud dog. This imported dog has gone on to be influential within the U.K.breeding population.
There have been sporadic reports of Irish Red and White Setters dying with symptoms consistent with CLAD. The pedigrees of several of these individuals have been traced back to one particular Irish Red and White Setter whose parentage may have included an Irish Setter. This dog then went on to sire two Red and White Setters which were widely used in the early breeding programmes. These two individuals are likely to have been carriers of the CLAD mutation, as affected offspring have been produced from litters where they occur on both sides of the pedigree. This strongly suggests that CLAD is apparent in at least one line of the Irish Red and White Setter population in the UK.
Here, we present evidence for the occurrence of the CLAD mutation in Irish Red and White Setters, and propose that DNA testing be undertaken before breeding programmes are instigated in lines at risk of CLAD.
Materials and Methods
Genomic DNA was extracted from 76 Irish Red and White Setter whole blood samples using a Nucleon kit ( Nucleon Biosciences, Coatbridge, UK). The CLAD CD18 mutation site was amplified using the following primers for the polymerase chain reaction (PCR): CLADF (5'-CTTCCCTGCCCCATATCC-3'), CLADR (5'CTCACGTCCTGTGGTCCC-3'). Each 20ml reaction consisted of 10pmol forward and reverse primer, 1.5mM magnesium chloride, 50ng DNA, 2.5 units AmpliTaq Gold (Perkin-Elmer, Foster City, CA), 10% DMSO, 125nM dNTPs, 1x Buffer A (PCR Optimiser Kit, Invitrogen BV, Groningen, The Netherlands) and deionized water. PCR was performed at the following cycle conditions: 95°C for 10 min, then 32 cycles of 95°C for 45 s, 55°C for 45 s, 72°C for 45 s. Amplified PCR product was purified using a Microcon YM100 Spin Column (Amicon Bioseparations, Millipore, Bedford, MA, USA) and 3ml run on a 3% agarose gel with DNA Quantification Standards (GibcoBRL Life Technologies, Paisley, UK). Products were sequenced using 1.6pmol CLADF primer and a dRhodamine Terminator Sequencing kit (Perkin-Elmer) according to the manufacturer's instructions. After ethanol precipitation, 4ml ABI loading buffer (containing 5:1 deionized formamide : 25mM EDTA with 50mg/ml Blue Dextran) was added to the samples. Samples were heat denatured at 95°C for 2 min before being placed on ice and loaded onto a 4.25% acrylamide denaturing gel for electrophoresis on an ABI 377 DNA Sequencer (Perkin-Elmer). CLAD status was determined using ABI Sequence Analysis Software ( Perkin-Elmer), allowing identification of the mutation site at position 137 in the 441bp PCR product. This is illustrated in Fig.1.
Out of 76 Irish Red and White Samples screened for the CLAD mutation, 6 carriers
were identified, the remainder exhibiting a normal genotype. The estimated disease allele frequency of approximately 4% closely compares with the 5% estimate for the Irish Setter population (Kijas et al, 1999). However, the Irish Red and White samples used here were not a random collection, as they were selected for a study on posterior polar cataract and hence may not be a true representation of the breed.
The pedigrees of the six carrier Irish Red and White Setters were examined to identify common ancestors likely to be the source of the disease in the breed. All of the carrier animals identified in this study trace back to a common founder individual. Due to the limited number of Irish Red and White Setters that have been used to form the population in the UK, CLAD testing would be of significant benefit for the healthy continuation of this breed.
Since the diagnostic CLAD test has become commercially available in the UK, the test has changed from the original oligo ligation assay (Kijas et al.,1999) to the more robust DNA sequencing test described here. This allows an effective service to be provided to breeders and is an important development in health screening for both the Irish Setter and Irish Red and White Setter populations.
Help and support of the Irish Red and White Setter and Irish Setter dog owners was given throughout this study. Work at the Animal Health Trust is supported by the Guide Dogs for the Blind Association. We also thank Dr J. Sampson and Diana Nicholson from the Kennel Club for the provision of pedigree information.
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