Leukocyte adhesion deficiency syndrome: a controversy solved.

Sadly, mistakes of nature are often our greatest learning tools. Such is the case with the recent description by three independent groups of the molecular etiology of leukocyte adhesion deficiency (LAD) syndrome III.1–3 The discovery that mutations in kindlin-3 are responsible for this rare genetic disorder teaches us a great deal about how leukocytes regulate adhesion and trafficking through their primary adhesive receptors—the b1-, b2and b3-integrin receptors. This story also teaches us how mistakes can be made (and avoided) in science. There are three types of LAD syndromes, LADI, II and III (also referred to as variant LADI).4 As the name implies, these diseases are characterized by an inability of leukocytes to adhere and migrate during inflammatory and host defense reactions. As a result, patients with these autosomal recessive syndromes present with repeated bacterial and fungal infections in the absence of pus formation. Hundreds of LADI patients have been described worldwide, all with mutations in the gene encoding the b2 leukocyte integrin. Less than 10 patients have been described with LADII, which is a clinically milder form of the disease, and is caused by mutations in a fucose transporter protein leading to poor formation of Sialyl Lewis X (CD15), the ligand for L-selectin. LADIII is also rare (approximately 20 patients worldwide, with the largest kindred of Turkish origin), but presents with both the immunodeficiency of LADI as well as severe bleeding disorders resembling Glanzman’s thrombasthenia—a known deficiency of the platelet aIIbb3-integrin. Unlike LADI, the characterized LADIII patients have normal cell surface expression of all the major leukocyte integrins; however, these receptors fail to become activated to mediate leukocyte and platelet adhesion and cell spreading. Hence, these patients must have a defect in the intracellular signaling pathways that regulate integrin activation. The signaling networks that regulate b1-, b2and b3-integrin function are complex.5 In resting leukocytes and platelets, the integrins are held in a bent/inactive conformation that limits their ability to interact with ligands (endothelial ICAMs or extracellular matrix proteins such as fibrinogen or collagen). This conformation is maintained by a salt bridge between the cytoplasmic tails of the a and b chains of the integrin heterodimer, and through association with proteins such as talin. Following cellular activation (by agonists including chemokines, growth factors, antigens or selectin ligands), the cytoplasmic tails of the integrins separate, transmitting a conformational change throughout the integrin heterodimer that results in unfolding of the extracellular domains to allow high-affinity ligand binding. The processes that mediate integrin activation are referred to as ‘inside–out’ signaling (detailed in Figure 1). The fact that LADIII patients fail to activate high-affinity integrin ligand binding led Alon et al.7 to focus on each of the molecules involved in the inside–out pathway. Initial reports suggested that Rap1 failed to become activated in leukocytes from several patients. The group investigated the upstream regulator of Rap1, CalDAG-GEF1, and in fact they identified a potential mutation in a splice acceptor site for exon16 of the CalDAGGEF1 gene in two of the LADIII patients.6 Leukocytes from these patients had low levels of CalDAG-GEF1 mRNA and the protein was not detected in cell lysates. The patients showed complete failure to activate b1 and b2-integrins on leukocytes, as well as b3integrins on platelets. Altogether, this explains the full clinical spectrum of infections and bleeding suffered by these children.8 The fact that CalDAG-GEF1-deficient mice manifest an overall syndrome extremely similar to human LADIII9 buttressed this group’s assertion that LADIII is caused by loss of CalDAG-GEF1 leading to impaired Rap1 activation and failure to activate leukocyte integrins. This view was reflected in a number of reviews of integrin signaling, including our own.5 Yet, when more LADIII patients were examined, in particular patients from other families, impairment of Rap1 activation and reduced CalDAG-GEF1 expression was not observed.3,10 Whereas some of these patients had the splice acceptor ‘mutation’ described by Alon’s group, others did not. These disparate results initiated the controversy as to the molecular nature of LADIII syndrome. To identify the genetic defect in these patients Kuijpers et al.1 carried out a complete homozygosity mapping experiment on a larger number of LADIII samples, using single nucleotide polymorphism oligonucleotide arrays. Although these experiments localized the causative genetic mutation to a region containing the CalDAG-GEF1 gene, not all patients had the splice acceptor mutation and no defects in CalDAG-GEF1 expression were found. Instead mutations were mapped to the FERMT3 gene, which encodes kindlin-3, located a mere 500 kb away from the CalDAG-GEF1 gene. Mutations creating premature stop codons (Arg509X, Arg573X and Trp229X) were found in the different families. Leukocytes from patients with these mutations lacked kindlin-3 protein by immunoblotting. These results appear to rule out CL Abram and CA Lowell are at the Department of Laboratory Medicine, University of California, San Francisco, CA, USA. E-mail: [email protected] Immunology and Cell Biology (2009), 1–3 & 2009 Australasian Society for Immunology Inc. All rights reserved 0818-9641/09 $32.00