Leukocyte adhesion deficiency (LAD) in humans is an autosomal recessive disorder characterized by severe recurrent infections, delayed umbilical cord separation, impaired wound healing, severe gingivitis, persistent leucocytosis, and absent pus formation (Anderson et al., "The Severe and Moderate Phenotypes of Heritable Mac-1, LFA-1 Deficiency: Their Quantitative Definition and Relation to Leukocyte Dysfunction and Clinical Features," J. Infect. Dis., 152:668-689 (1985); Guneser et al., "An Infant With Severe Leucocyte Adhesion Deficiency," Acta Pediatr., 85:622-624 (1996)). The clinical manifestations of LAD are caused by diminished or absent expression of the .beta.-2 integrin (CD18) molecule (Arnaout et al., "Deficiency of a Leukocyte Surface Glycoprotein (LFAI) in Two Patients With Mol Deficiency." J. Clin. Invest., 74: 1291-1300 (1984), El Habbal et al., "Leucocyte Adhesion Deficiency," Archives of Disease in Childhood, 69:463-466 (1993)). Integrins are heterodimeric glycoproteins composed of an .alpha. and .beta. subunit and are known to play a central role in cell-cell adhesion events. The four leukocyte integrin .alpha. subunits .alpha.L (CD11a), .alpha.M (CD11b), .alpha.X (CD11c), and .alpha.D (CD11d) each associate with the common .beta.-2 subunit (CD18) to form the antigens LFA-1, Mac-1, p150,95, and .alpha..sub.D.beta..sub.2, respectively (Springer et al., "Inherited Deficiency of the Mac-1, LFA-1, p150,95 Glycoprotein Family and Its Molecular Basis," J. Exp. Med., 160:1901-1918 (1984); Danilenko et al., "A Novel Canine Leukointegrin, Alpha d Beta 2, Is Expressed by Specific Macrophage Subpopulations in Tissue and a Minor CD8+Lymphocyte Subpopulation in Peripheral blood, J. Immunol., 155:35-44 (1995)). Defects in the common .beta.2 subunit prevent surface expression of all four heterodimers, and result in the LAD phenotype.
The human .beta.-2 integrin gene (ITGB2) is composed of 16 exons spanning approximately 40 kb of genomic sequence (Weitzman et al., "The Gene Organization of the Human Integrin p2 Subunit (CD18)," FEBS, 294:97-103 (1991)). Mutation screening of ITGB2 has revealed a spectrum of mutations in human LAD patients, the majority of which are missense mutations changing residues in a highly conserved region of the extracellular domain (Arnaout et al., "Point Mutations Impairing Cell Surface Expression of the Common Beta Subunit (CD 18) in a Patient with Leukocyte Adhesion Molecule (Leu-CAM) Deficiency." J. Clin. Invest., 85:977-981 (1990); Wardlaw et al., "Distinct Mutations in Two Patients with Leukocyte Adhesion Deficiency and Their Functional Correlates," J. Esp. Med., 172:335-345 (1990); Matsuura et al., "Leukocyte Adhesion Deficiency: Identification of Novel Mutations in Two Japanese Patients with a Severe Form," Biochem. Biophys. Res. Commun., 184:1460-1467 (1992)). In addition, a mutation at the initiation codon (Sligh et al., "An Initiation Codon Mutation in CD18 in Association with the Moderate Phenotype of Leukocyte Adhesion Deficiency," J. Biol. Chem., 267:714-718 (1992)), frameshift mutations (Back et al., "Identification of Two Molecular Defects in a Child with Leukocyte Adherence Deficiency," J. Biol. Chem., 267:5482-5487 (1992)), and splice mutations (Nelson et al., "Genetic Cause of Leukocyte Adhesion Molecule Deficiency: Abnormal Splicing and a Missense Mutation in a Conserved Region of CD18 Impair Cell Surface Expression of Beta-2 Integrins," J. Biol. Chem., 267:3351-3357 (1992)) have been shown to result in LAD. On the basis of very similar clinical symptoms, leukocyte .beta.2 integrin expression was examined and found to be defective in American Holstein-Friesian cattle with a severe immunodeficiency syndrome (Kehrli et al., "Molecular Definition of the Bovine Granulocytopathy Syndrome: Identification of Deficiency of the Mac-1 (CD11b/CD 18) Glycoprotein." Am. J. Vet. Res., 51:1826-1836 (1990)). Sequence analysis of bovine ITGB2 revealed that all affected calves tested were homozygous for a missense mutation (D128G). Thus, the bovine form of the disease (BLAD) is genetically equivalent to that of human LAD (Shuster et al., "Identification and Prevalence of a Genetic Defect that Causes Leukocyte Adhesion Deficiency in Holstein Cattle," Proc. Natl. Acad. Sci. USA, 89:9225-9229 (1992))
Renshaw et al., "Canine Granulocytopathy Syndrome: Neutrophil Dysfunction in a Dog with Recurrent Infections," J. Am. Vet. Med. Assoc., 166:443-447 (1975) described a case of granulocytopathy with impaired leukocyte bactericidal activity and life-threatening infections in an Irish Setter. Subsequent breeding experiments demonstrated an autosomal recessive nature of the defect (Renshaw et al., "Canine Granulocytopathy Syndrome: An Inherited Disorder of Leukocyte Function," Am. J. Pathol., 95,731 -743 (1979)). Deficient expression of the CD11/CD18 molecule was subsequently identified in an animal with recurrent infections (Giger et al., "Deficiency of Leukocyte Surface Glycoproteins Mol, LFA-1, and Leu M5 in a Dog with Recurrent Bacterial Infections: An Animal Model," Blood, 69:1622-1630 (1987)). Further, defective adhesion and C3b medicated phagocytosis were demonstrated in 12 Irish Setter puppies with severe infections of omphalophlebitis, skin infections, osteomyelitis, and gingivitis (Trowald-Wigh et al., "Leucocyte Adhesion Protein Deficiency in Irish Setter Dogs," Veterinary Immunology and Immunopathology, 32:261-380 (1992)), providing a final confirmation of the existence of canine leukocyte adhesion deficiency (CLAD). CLAD is a fatal immunodeficiency disease so far only found in Irish Setters. To date, the molecular defect responsible for CLAD is unknown, making molecular identification of carrier animals impossible.
The present invention is directed to overcoming the above-noted deficiencies in the prior art.