Hematologically important mutations: Leukocyte Adhesion Deficiency (first update)

Introduction

Leukocyte adhesion deficiency (LAD) is an autosomal recessive disorder caused by decreased expression or functioning of CD18, the β₂ subunit of the leukocyte β₂ integrins [1]. This deficiency leads to severe impairment of leukocyte adhesion to the vascular wall and leukocyte migration to sites of infection and inflammation. The patients suffer from recurrent, life-threatening bacterial and fungal infections and from impaired wound healing. Characteristic features are delayed separation of the umbilical cord and strong leukocytosis, especially neutrophilia, during periods of infection. Many LAD patients die at young age despite intensive antibiotic therapy. Bone-marrow transplantation is the treatment of choice. LAD is a rare immunodeficiency, but the exact incidence is not known.

The integrins are transmembrane receptors composed of α and β subunits that mediate cellular adhesive interactions throughout the body. At present 18 α and 8 β subunits have been identified that are loosely organized into integrin families. The β₂ integrins form a family of four heterodimeric proteins, only expressed on leukocytes, with one of four α subunits coupled to a common β₂ subunit: α_Lβ₂ (LFA-1, CD11a/CD18), α_Mβ₂ (Mac-1 or CR3, CD11b/CD18), α_Xβ₂ (p150,95, CD11c/CD18) and α_Dβ₂ (CR4, CD11d/CD18), the latter only being expressed on macrophages. Decreased expression of the common β₂ subunit leads to a similar decrease in the expression of all four α subunits on the leukocyte surface. The four β₂ integrins act as adhesion proteins, mediating adhesion of leukocytes to other cells and to extracellular matrix proteins. The α subunits and the β₂ subunit are transmembrane proteins, intracellularly connected to the leukocyte cytoskeleton. Binding to extracellular ligands leads to a conformational change of the β₂ integrins, increased binding of intracellular target proteins and downstream signal transduction to cell spreading and altered gene expression, cell proliferation, differentiation and apoptosis (“outside-in” signaling). Leukocyte activation, e.g. as a result of chemokine binding to chemokine receptors, antigen binding to the T-cell receptor or ligand binding to selectins, induces conformational changes in the extracellular regions of the β₂ integrins, leading to a higher affinity for their ligands (“inside-out” signaling) [2].

Classical LAD-I

In the most common form of LAD, called LAD-I (OMIM #116920), mutations are found in ITGB2 (integrin beta-2), the gene located at 21q22.3 (OMIM *600065) that encodes the β₂ integrin protein. Usually, this leads to the absence or decreased expression of the β₂ integrins on the leukocyte surface, but sometimes a normal expression of nonfunctional β₂ integrins is found. In a previous publication we listed 34 mutations found in ITGB2 of LAD-I patients [3]. In the present publication, 53 newly identified mutations have been added to Table 1 (marked with * in the last column). Mutations that have not been previously published elsewhere are marked as “Unpubl.”. Most of the point mutations are found in a ~240-residue domain that is highly conserved in all β integrin subunits and coded for by exons 5 – 9 of ITGB2 (figure 1). This “βI domain”, together with its αI counterpart, constitutes the major ligand-binding site of the β₂ integrins. Both I domains also contain a metal ion-dependent adhesion site (MIDAS motif) consisting of an Asp-X-Ser-X-Ser sequence.

Table 2 contains information on apparently benign polymorphisms that have been identified in ITGB2. The mutation c.1756C>T (p.Arg586Trp) in ITGB2 was found in two non-related patients on the same allele with the mutation c.742-14C>A at the 3’ end of intron 6 (leading to the incorporation of four additional amino acids in exon 7) [4,5]. By itself, the 586Trp CD18 molecule, expressed in COS cells, supports about 75% of normal β₂ integrin expression [5], indicating that this mutation may be regarded as a polymorphism. Therefore, the c.1756G/T alternatives are shown as SNPs in Table 2. Other polymorphisms shown in Table 2 have not yet been defined at the functional level.

LAD-II

Two other, extremely rare forms of LAD exist. Patients with LAD-II (OMIM #266265) have a defect in the fucosylation of various cell surface glycoproteins, some of which function as ligands for L-selectin [6]. As a result, the initial “rolling” of leukocytes over the endothelial vessel wall in areas of inflammation, which is mediated by reversible contact between L-selectins on the leukocytes and E- or P-selectins on the endothelial cells with their respective sialated fucosyl ligands on the opposite cells, is disturbed. Both via intracellular signaling and by slowing down the leukocytes, this rolling allows integrins to bind their ligand on endothelial cells, which is needed for stable adhesion. Thus, in LAD-II, one mechanism of β₂-integrin activation is lacking, leading to decreased leukocyte adhesion to the vessel wall and decreased transendothelial migration into the tissues. However, the chemokine and T-cell receptor pathways of β₂-integrin activation are still operative, and the infectious episodes in LAD-II patients are therefore in general less severe than those seen in LAD-I patients. On the other hand, the fucosylation defect affects not only selectin ligands but also other essential glycoproteins, leading to severe mental and growth retardation. The molecular defect in LAD-II has been identified as a deficiency in a GDP-fucose transport protein in the Golgi system [7,8]. This protein is encoded by SLC35C1 (Solute carrier family 35 member C1) at 11p11.2 (OMIM *605881). Table 3 lists the mutations found in this gene in six families with LAD-II patients. Two of these mutations concern Arg147 and Thr308, both highly conserved amino acids in the family of nucleotide-sugar transporters group 2 and suggested to be involved in substrate recognition [9]. Supplementation of fucose led to a substantial clinical improvement and correction of hypofucosylation in the patient homozygous for the Arg147Cys mutation, whereas it was of no benefit to the patients homozygous for the Thr308Arg mutation [7,8]. Possible polymorphisms in SLC35C1 are also listed in Table 3.

LAD-III (LAD-I/variant)

Finally, patients have been described with a defect in the “inside-out” signaling of leukocytes required for activation of β₂ integrins into structures that bind their ligands with high affinity [10]. These patients, in addition to infections, also present with a bleeding disorder, indicating that the signaling defect also affects the β₃ integrin fibrinogen receptor α_IIbβ₃ on blood platelets. The molecular defect of this variant form of LAD (LAD-III or LAD-I/variant, OMIM #612840) has recently been assigned to mutations in FERMT3 (fermitin family homolog 3) at 11q13.1 (OMIM *607901), the gene encoding kindlin-3, a protein involved in inside-out signaling to all blood cell-expressed β integrins (β₁, β₂ and β₃) [11-14]. Kindlin-3 is apparently expressed not only in hematopoietic cells, but also in endothelial cells [15]; the biological significance of its deficiency in endothelial cells in LAD-III is not yet clear. A discussion has raged in the literature about the importance of a genetic variation in the gene encoding CalDAG-GEF1 (a guanine nucleotide exchange factor for Rap1, involved in integrin activation) in some patients with LAD-III, in addition to mutations in FERMT3 found in these patients [11,12,16]. However, since the functional defect in such patients can be corrected by reconstitution with kindlin-3 but not by reconstitution with CalDAG-GEF1 [14], this variation in CalDAG-GEF1 is of no importance for the functional defect in LAD-III patients. Table 4 and figure 1 list the mutations found in FERMT3 in 21 families with LAD-III patients. A hotspot of p.Arg509X mutations in kindlin-3 points to a founder effect, since these mutations are all found in Turkish families originating from Anatolia.

Final remarks

Additional information about the tabulated mutations and about LAD in general can be found in a recent review [16] and in the cited literature. In Table 1 we have used the notation LAD-I^o, LAD-I⁻ and LAD-I⁺ for differentiating the various phenotypes of LAD-I. In this nomenclature the superscript symbol indicates whether the protein is present at < 5% of normal expression (^o), diminished in expression (⁻), i.e. between 5% and 20% of normal expression, or normally present but nonfunctional (⁺).This information is based on immune reactivity of the patients’ leukocytes with monoclonal antibodies analyzed by flow cytometry and sometimes on similar analyses of COS cells cotransfected with mutant CD18 molecules and wild-type CD11 molecules. In case this information is not known, this is indicated as (^?). In a number of cases functionality of the mutant CD18 proteins was tested in cellular adherence assays to β₂ ligands.

The nucleotide numbering system we have used is based on the cDNA sequence and follows the convention that +1 is the A of the ATG initiation codon. This differs from the numbering of the GenBank sequences; for ITGB2 (GenBank Accession Number M15395) subtract 72 from the GenBank sequence number to make the initiator A +1. The notation of the mutations follows the recommendations of the Human Genome Variation Society [17] (see also www.hgvs.org/mutnomen). The consequences of the mutations for protein composition have been checked with the Mutalyzer program (www.lovd.nl/mutalyzer) [18]. Where possible we have cross-referenced the mutations indicated in the present article with those in an LAD-I, LAD-II and LAD-III database that lists these patients by accession number. These databases contain additional biochemical, genetic and clinical information and are available at www.uta.fi/imt/bioinfo/ITGB2base, www.uta.fi/imt/bioinfo/SLC35C1base and www.uta.fi/imt/bioinfo/FERMT3base, respectively. Moreover, information can also be found in the HGMD database at www.hgmd.cf.ac.uk/ac/search.php.