Canine leucocyte adhesion deficiency in Irish red and white setters

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Canine leucocyte adhesion deficiency in Irish red and white setters Seventy-six Irish red and white setter samples were tested for the recently documented CD18 point mutation which manifests as canine leucocyte adhesion deficiency (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 is the first demonstration that the CLAD mutation exists outside the Irish setter population, confirming that the mutation is present in a significant minority of Irish red and white setters. Routine testing by breeders to identify carrier animals will enable the eradication of CLAD from the Irish red and white setter population. S. L. DEBENHAM, A. MILLINGTON*, J. KIJAS†, L. ANDERSSON† AND M. BINNS Journal of Small Animal Practice (2002) 43, 74–75

Animal Health Trust, Lanwades Park, Kentford, Newmarket, Suffolk CB8 7UU *Genetic Sub-Committee, Irish Red & White Setter Club of Great Britain †Department of Animal Breeding and Genetics, Swedish University of Agricultural Sciences, Box 597, S-751 24 Uppsala, Sweden

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INTRODUCTION Leucocyte 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 the condition are caused by deficient expression of the -2 integrin (CD18) molecule (Arnaout and others 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, manifested with very similar symptoms to those in humans (Kehrli and others 1990). Molecular studies proved that these symptoms were also caused by defective -2 integrin expression, due to a missense mutation within the coding region of the gene (Shuster and others 1992). Canine leucocyte adhesion deficiency (CLAD) in Irish setters was first identified in 1975 (Renshaw and others 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 and others 1992). The identification of the gene mutation responsible for the disease in Irish setters was ascertained after mutational analysis of CD18 in Irish setter CLAD pedigrees (Kijas and others 1999). From this, a single missense mutation – Cys36Ser – was identified, which showed complete association with CLAD in the breed (Kijas and others 1999). The mutation is thought to be responsible for incomplete disulphide bonding within the -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. Analysis of over 300 dogs identified carriers across Europe and extending to the southern hemisphere (Australia), indicating that a substantial radiation of the disease allele has occurred (Kijas and others 2000). The Irish red and white setter breed is derived from the same roots as the Irish red setter breed. Originally, in Ireland, only pure Irish red setters were used as show and working dogs. 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. 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 Irish 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

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A

B

FIG 1. Molecular distinction between normal and carrier animals of CLAD. (A) A CLAD carrier, possessing one mutant base (C) and one normal base (G) for the CLAD mutation site. The letter S is the designated IUB code used to represent the C/G heterozygous base position. (B) The DNA sequence of an animal with a normal genotype (GG) for the CLAD mutation site within the CD18 molecule

strongly suggests that CLAD is apparent in at least one line of the current Irish red and white setter population in the UK. This paper presents evidence for the occurrence of the CLAD mutation in Irish red and white setters, and the authors propose that DNA testing is undertaken before breeding programmes are instigated.

MATERIALS AND METHODS Genomic DNA was extracted from 76 Irish red and white setter whole blood samples using a Nucleon kit (Nucleon Biosciences, Coatbridge). The CLAD CD18 mutation site was amplified using the following primers for the polymerase chain reaction (PCR): CLADF (5-CTTCCCTGCCCCATATCC-3) and CLADR (5CTCACGTCCTGTGGTCCC-3). Each 20 µl reaction consisted of 10 pmol forward and reverse primer, 1·5 mM magnesium chloride, 50 ng DNA, 2·5 units AmpliTaq Gold (Perkin-Elmer, Foster City, CA), 10 per cent DMSO, 125 nM dNTPs, 1Buffer A (PCR Optimiser Kit, Invitrogen BV, Groningen, The Netherlands) and deionised water. PCR was performed at the following cycle conditions: 95°C for 10 minutes, then 32 cycles of 95°C for 45 seconds, 55°C for 45 seconds and 72°C for 45 seconds. Amplified PCR product was purified using a Microcon YM100 Spin Column (Amicon Bioseparations, Millipore, Bedford, MA, USA) and 3 µl was run on a 3 per cent agarose gel with DNA Quantification Standards (GibcoBRL Life Technologies, Paisley). Products were sequenced using 1·6 pmol forward primer and a dRhodamine Terminator Sequencing kit (Perkin-Elmer), according to the manufacturer’s instructions. After ethanol precipitation, 4 µl ABI loading buffer (containing 5:1 deionised formamide:25 mM EDTA with 50 mg/ml JOURNAL OF SMALL ANIMAL PRACTICE

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Blue Dextran) was added to the samples. Samples were then heat denatured at 95°C for two minutes before being placed on ice and loaded on to a 4·25 per cent 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 441 base pair PCR product. This is illustrated in Fig 1.

RESULTS AND DISCUSSION Out of 76 Irish red and white setter samples screened for the CLAD mutation, six carriers were identified; the remainder exhibited a normal genotype. The estimated disease allele frequency of approximately 4 per cent in this breed compares closely with the 5 per cent estimated for the Irish setter population (Kijas and others 1999). However, the Irish red and white setter samples used in the present study 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 future health of this breed. Since the diagnostic CLAD test became commercially available in the UK, the test has been changed from an oligoligation assay (Kijas and others 1999) to more robust DNA sequencing tests, as described here, or based on the pyrosequencing method (Kijas and others 2000). These tests both allow a

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reliable service to be provided to breeders and represent an important development in health screening for both the Irish setter and Irish red and white setter populations. Acknowledgements Help and support from the owners of the Irish red and white setters and Irish setters studied was given throughout this investigation. The authors thank Peter James and Rita Bryden for their helpful comments on the manuscript. Work at the Animal Health Trust is supported by the Guide Dogs for the Blind Association. The authors also thank Dr J. Sampson and Diana Nicholson, of the Kennel Club, for the provision of pedigree information. References ARNAOUT, M. A., SPITS, H., TERHORST, C., PITT, J. & TODD, R. F. (1984) Deficiency of a leukocyte sur face glycoprotein (LFA1) in two patients with Mo1 deficiency. Journal of Clinical Investigations 74, 1291-1300 EL HABBAL, M. H. & STROBEL, S. (1993) Leucocyte adhesion deficiency. Archives of Disorders in Childhood 69, 463-466 KEHRLI, M. E. JR, SCHMALSTIEG, F. C., ANDERSON, D. C., VAN DER MAATEN, M. J., HUGHES, B. J., ACKERMANN, M. R., WILHELMSEN, C. L., BROWN, G. B., STEVENS, M. G. & WHETSTONE, C. A. (1990) Molecular definition of the bovine granulocytopathy syndrome: identification of deficiency of the Mac-1 (CD11b/CD18) glycoprotein. American Journal of Veterinary Research 51, 18261836 KIJAS, J. M. H., BAUER, T. R. JR, GÄFVERT, S., MARKLUND, S., TROWALD-WIGH, G., JOHANNISSON, A., HEDHAMMAR, Å., BINNS, M., JUNEJA, R. K., HICKSTEIN, D. D. & ANDERSSON, L. (1999) A missense mutation in the -2 integrin gene (ITGB2) causes canine leucocyte adhesion deficiency. Genomics 61, 101-107 KIJAS, J. M. H., JUNEJA, R. K., GAFVERT, S. & ANDERSSON, L. (2000) Detection of the causal mutation for canine leucocyte adhesion deficiency (CLAD) using pyrosequencing. Animal Genetics 31, 326-328 RENSHAW, H. W., CHATBURN, C., BRYAN, G. M., BARTSCH, R. C. & DAVIS, W. C. (1975) Canine granulocytopathy syndrome: neutrophil dysfunction in a dog with recurrent infections. Journal of the American Veterinary Medical Association 166, 443-447 RENSHAW, H. W. & DAVIS, W. C. (1979) Canine granulocytopathy syndrome: an inherited disorder of leukocyte function. American Journal of Pathology 95, 731-743 SHUSTER, D. E., KEHRLI, M. E. JR, ACKERMANN, M. R. & GILBERT, R. O. (1992) Identification and prevalence of a genetic defect that causes leukocyte adhesion deficiency in Holstein cattle. Proceedings of the National Academy of Sciences USA 89, 92259229 TROWALD-WIGH, G., HÅKANSSON, L., JOHANNISSON, A., NORRGREN, L. & HÅRD AF SEGERSTAD, C. (1992) Leucocyte adhesion protein deficiency in Irish Setter dogs. Veterinary Immunology and Immunopathology 32, 261-280

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