WHIM syndrome caused by a single amino acid substitution in the carboxy-tail of chemokine receptor CXCR4

Abstract

WHIM syndrome is a rare, autosomal dominant, immunodeficiency disorder so-named because it is characterized by warts, hypogammaglobulinemia, infections, and myelokathexis (defective neutrophil egress from the BM). Gain-of-function mutations that truncate the C-terminus of the chemokine receptor CXCR4 by 10-19 amino acids cause WHIM syndrome. We have identified a family with autosomal dominant inheritance of WHIM syndrome that is caused by a missense mutation in CXCR4, E343K (1027G → A). This mutation is also located in the C-terminal domain, a region responsible for negative regulation of the receptor. Accordingly, like CXCR4(R334X), the most common truncation mutation in WHIM syndrome, CXCR4(E343K) mediated approximately 2-fold increased signaling in calcium flux and chemotaxis assays relative to wild-type CXCR4; however, CXCR4(E343K) had a reduced effect on blocking normal receptor down-regulation from the cell surface. Therefore, in addition to truncating mutations in the C-terminal domain of CXCR4, WHIM syndrome may be caused by a single charge-changing amino acid substitution in this domain, E343K, that results in increased receptor signaling.

Figure 1

Clinical manifestations in a family with WHIM syndrome. (A) Pedigree consistent with autosomal-dominant inheritance. (B) Representative area of skin warts on patient P1. Arrows point to clusters of representative warts on the dorsum of the right hand. (C) BM biopsy from P1 showing full myeloid maturation in the face of peripheral neutropenia, which is consistent with myelokathexis (400×). Arrows point to representative mature neutrophils and granulocyte precursors and an eyeglass-shaped neutrophil in which pyknotic nuclear lobes were connected by a long filament.

Figure 2

CXCR4^E343K is associated with WHIM syndrome. (A) Position of mutation E343K in the 7-transmembrane domain model of CXCR4. Small dots represent amino acids except serine (designated by S). The locations of other point mutations reported in WHIM syndrome are also highlighted. (B) Alignment of predicted amino acid sequences from the C-terminal domain of CXCR4^WT and all reported CXCR4 mutants associated with WHIM syndrome. The position mutated to lysine (K) in CXCR4^E343K is highlighted. *Missense mutation that extends the sequence by an additional 10 amino acids, not shown for clarity. (C) The glutamic acid changed to lysine in CXCR4^E343K is highly conserved phylogenetically. Predicted amino acid sequences from the C-terminal domain of CXCR^WT are aligned for the indicated species. The homology (percent amino acid identity) of human CXCR4 to CXCR4 from each of the other species listed is shown for both the full-length sequence and the C-terminal 19 amino acids.

Figure 3

CXCR4^E343K is a gain-of-function mutation: analysis in transfected K562 cells. (A,C) Expression of CXCR4 variants on untransfected K562 cells (gray area) or K562 cells transfected transiently (A) or stably (C) with CXCR4^WT (solid line), CXCR4^R334X (dashed line), and CXCR4^E343K (dotted line). X-axis shows the mean fluorescence intensity of CXCR4 expression. (B) Peak calcium-flux responses of K562 cells transiently transfected with CXCR4^WT, CXCR4^R334X, and CXCR4^E343K in response to the indicated doses of CXCL12. *P < .05; **P < .01; and ***P < .001 for the comparison between CXCR4^WT and either CXCR4^R334X or CXCR4^E343K. Data are summarized as the means ± SEM from 4 independent experiments with 3 replicates each. (D) The peak calcium-flux response of K562 cells stably transfected with CXCR4^WT, CXCR4^R334X, and CXCR4^E343K in response to 50nM CXCL12 stimulation. *P < .05 and **P < .01 for the comparison between CXCR4^WT and CXCR4^E343K or CXCR4^R334X.

Figure 4

CXCR4^E343K is a gain-of-function mutation: analysis in primary leukocytes from patients with WHIM syndrome. (A-B) CXCR4 expression on PBMCs. Cells were gated on lymphocytes and monocytes. Representative zebra plots are shown in panel A and summary data in panel B. Percentages of CXCR4⁺ and CXCR4⁻ cells in PBMCs are shown. (C) PBMC calcium-flux responses to 10nM CXCL12. (D) Patient and HD PBMC calcium-flux responses to CXCL12 can both be blocked by plerixafor. CXCL12 was added after 3 minutes of incubation with the indicated concentrations of plerixafor. (E) Representative CXCR4 expression on neutrophils from an HD and from P1. Shaded area is unstained; solid line, CXCR4; dashed line, isotype control. (F) Neutrophil chemotaxis. Comparisons are displayed of a HD versus P1 and P2 for different concentrations of CXCL12-induced chemotaxis. Chemotactic index was defined as 1 when no CXCL12 was added to the lower chamber. Results from both patients were averaged and graphed, with SEM displayed for each value. *P < .05; **P < .01; and ***P < .001 for the comparison between HDs and CXCR4^E343K (E343K) patients, respectively.

Figure 5

WHIM mutation CXCR4^E343K attenuates CXCL12-induced receptor down-regulation but to a lesser degree than CXCR4^R334X. Surface expression was measured with CXCR4-specific mAb 12G5 for the cell types indicated at the top of each panel after incubation in the presence or absence of the stimuli indicated on the x-axis. (A) Transiently transfected K562 cells. **P < .01 and ***P < .001 for the comparison between CXCR4^WT (WT) and CXCR4^E343K (E343K) or CXCR4^R334X (R334X), respectively. Data are summarized as the means ± SEM from 4 experiments. (B) PBMCs. Data are pooled from multiple independent experiments with cells with the indicated CXCR4 genotypes (number of individuals studied for each genotype). P1-P3 were studied twice and P4 once; 1 CXCR4^R334X patient was studied twice and the other 2 once, and each HD was studied once. One or 2 HDs were studied as controls in each of the 5 experiments involving WHIM patients, and the rest were studied in 8 additional independent experiments. Data are the means ± SEM of all values in each category. *P < .05; **P < .01; and ***P < .001 for the comparison between HDs and CXCR4^R334X (R334X) or CXCR4^E343K (E343K) patients, respectively. (C) B cells. After incubation with stimuli in duplicates, PBMCs were gated on CD19⁺ cells and analyzed for CXCR4 expression. Data are from 1 experiment that included WHIM patients P1-P4, 1 CXCR4^R334X patient, and 1 HD, and 2 additional independent experiments for the other 4 HDs, and are the means ± SEM of all values in each category. **P < .01 and ***P < .001 for the comparison between HDs and CXCR4^R334X (R334X) or CXCR4^E343K (E343K) patients, respectively. (D) Normal homologous desensitization of WHIM variants CXCR4^R334X (R334X) and CXCR4^E343K (E343K) expressed in K562 cells. Data were pooled from 5 independent experiments done in triplicate with the indicated variants and are summarized as the means for each condition.