Leukocyte adhesion deficiency-III is caused by mutations in KINDLIN3 affecting integrin activation

Abstract

Integrins are the major adhesion receptors of leukocytes and platelets. Beta1 and beta2 integrin function on leukocytes is crucial for a successful immune response and the platelet integrin alpha(IIb)beta3 initiates the process of blood clotting through binding fibrinogen. Integrins on circulating cells bind poorly to their ligands but become active after 'inside-out' signaling through other membrane receptors. Subjects with leukocyte adhesion deficiency-1 (LAD-I) do not express beta2 integrins because of mutations in the gene specifying the beta2 subunit, and they suffer recurrent bacterial infections. Mutations affecting alpha(IIb)beta3 integrin cause the bleeding disorder termed Glanzmann's thrombasthenia. Subjects with LAD-III show symptoms of both LAD-I and Glanzmann's thrombasthenia. Their hematopoietically-derived cells express beta1, beta2 and beta3 integrins, but defective inside-out signaling causes immune deficiency and bleeding problems. The LAD-III lesion has been attributed to a C --> A mutation in the gene encoding calcium and diacylglycerol guanine nucleotide exchange factor (CALDAGGEF1; official symbol RASGRP2) specifying the CALDAG-GEF1 protein, but we show that this change is not responsible for the LAD-III disorder. Instead, we identify mutations in the KINDLIN3 (official symbol FERMT3) gene specifying the KINDLIN-3 protein as the cause of LAD-III in Maltese and Turkish subjects. Two independent mutations result in decreased KINDLIN3 messenger RNA levels and loss of protein expression. Notably, transfection of the subjects' lymphocytes with KINDLIN3 complementary DNA but not CALDAGGEF1 cDNA reverses the LAD-III defect, restoring integrin-mediated adhesion and migration.

Figure 1

The CALDAGGEF1 gene C→A base change has no effect on mRNA and protein levels or on deficient LAD-III B cell adhesion and migration. (a) The DNA sequence surrounding the C→A base change in exon 16 of the CALDAGGEF1 gene in the Turkish (C→A base change) and Maltese (no base change) families. C or A indicates homozygosity; C/A indicates heterozygosity. The original data are shown in Supplementary Figure 1. (b) CALDAG-GEF1 protein abundance in subjects (S), relatives (M, mother; F, father) and control individuals (C), as assessed by western blotting (n = 2 independent samples per family). α-TUBULIN was used as a loading control. (c) Migration characteristics of LAD-III B cells adhered to ICAM-1. LAD-III cells from each family were transfected with a wild-type CALDAGGEF1 or EGFP cDNA construct; EGFP-transfected parents’ (families 1 and 3, mother; family 2, father) cells are shown for comparison. n = 4 independent experiments for each family. Data are shown as means ± s.e.m.; ***P < 0.001. (d) Left, differential interference contrast (DIC) and IRM images of B cells from the family 3 subject with LAD-III and from the mother (Parent) transfected as in c (n = 2). Scale bar, 10 μm. Right, quantification of the area of close contact for n = 35 cells per cell type. Data are shown as means ± s.e.m.; ***P < 0.001.

Figure 2

Mutations in the KINDLIN3 gene. (a) Top, the DNA sequence around the location of the C→T base change in exon 12 of the KINDLIN3 gene in the Turkish (mutated) and Maltese (not mutated) families; C or T indicates homozygosity; C/T denotes heterozygosity. Bottom, traces of the DNA sequence analysis around the mutation site in exon 12 for wild-type genome (WT), a heterozygote Turkish parent (HET) and a homozygous mutant Turkish subject with LAD-III (LAD-III). Y indicates the presence of a pyrimidine (both C and T). (b) Top, the DNA sequence around the location of the A→G base change in the KINDLIN3 exon 14 splice acceptor site in the Maltese (mutated) and Turkish (not mutated) families; A or G at this position indicates homozygosity; A/G denotes heterozygosity. Bottom, traces of the DNA sequence analysis around the mutation site in splice acceptor site of exon 14 for the WT, the heterozygote Maltese parents (HET) and the homozygous mutant Maltese subject with LAD-III; R indicates the presence of a purine (both A and G). More extensive original data for a and b are shown in Supplementary Figure 5 online. (c) Diagrammatical representation of human KINDLIN-3 protein (residues 1-663), showing the three FERM subdomains (F1-3) characteristically intersected with a pleckstrin homology (PH) domain. The location of the exons encoding the different regions of the KINDLIN-3 protein is shown. The position of the two LAD-III mutations, one in exon 12 (E12) and the other affecting the join of E13 and E14 of the KINDLIN3 gene, detailed above in a and b, respectively, are highlighted with red asterisks.

Figure 3

Analysis of mutated KINDLIN3 mRNA in subjects with LAD-III and their relatives. (a) Quantification of KINDLIN3 mRNA by quantitative RT-PCR analysis with probes covering exons 6-7 and 13-14. Subjects and their relatives were compared to two control EBV-transformed cell lines (set at 100%). n = 2 for each family; ***P < 0.001. Si, sibling. (b) Effect of the splice acceptor site mutation in the Maltese subject on KINDLIN3 mRNA at the exon 13-14 junction. The RT-PCR product encompassing the mRNA sequence specifying exons 12-15 was compared between control (WT) and the Maltese LAD-III subject. The unique DNA sequence specifying the end of exon 13 (last seven base pairs) and the start of exon 14 (25 base pairs) is shown for WT. For the Maltese subject with LAD-III, aberrant sequence is apparent after the last seven bases of exon 13 (arrow indicates beginning of aberrant sequence) which is consistent with an exon 14 splice acceptor mutation. (c) KINDLIN-3 protein abundance in each family, as detected in the B cells of the parents (M, F) but not of the LAD-III subjects (S). A nonrelated EBV transformed cell line (C) and primary T lymphoblasts (T) are included as controls. α-TUBULIN was used as a loading control.

Figure 4

Adhesion and migration characteristics of LAD-III B cells expressing wild-type Kindlin-3. (a) Left, DIC and IRM images of adhesion on ICAM-1 of LAD-III B cells transfected with either EGFP or a KINDLIN3 cDNA construct and compared with a parent’s B cells (family 1 and 3, mother; family 2, father) transfected with EGFP (n = 2 for each cell type). Right, quantification of the area of close contact for n = 35 cells. Scale bar, 10 μm. Data are shown as means ± s.e.m.; *, P < 0.05, ***P < 0.001 and NS, not significant. (b) Migration characteristics of LAD-III B cells adhered to ICAM-1. B cells from subjects with LAD-III were transfected with either EGFP or wild-type EGFP-KINDLIN3 cDNA constructs and compared with EGFP-transfected parents’ B cells (family 1 and 3, mother; family 2, father) (representative experiment of n = 3 for each family). Data are shown as means ± s.e.m.; **P < 0.01 and ***P < 0.001. (c) The cell-tracking results from an individual experiment with cells from the family two subject with LAD-III and from his father, transfected as in b.