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Table 2. Comparison of the clinical characteristics of 14 patients with CXCR4 gain-of-function mutations and four patients with CXCR2 loss- of-function mutations enrolled in the French Severe Congenital Neutropenia Registry.
Characteristic
Number of patients
Age at diagnosis (years) Age at last visit (years) Oral lesions
Severe infections
Warts
Hematologic values (all available CBC)
Neutrophils (x109/L)
Monocytes (x109/L)
Lymphocytes (x109/L)
Hemoglobin (g/dL)
Platelets (x109/L)
Myelokathexis
Bone-marrow myeloid/erythroid ratio G-CSF treatment
Immunoglobulin levels (g/L)
IgG IgM IgA
Lymphocyte subsets
CD3+CD4+ T cells (x109/L) CD3+CD8+ T cells (x109/L)
CD19+ B cells (x109/L) CD3–CD16+CD56+ NK cells (x109/L)
Solid tumors
Tetralogy of Fallot
Deaths
Patients with CXCR4 mutations
14
4.9 (0.1-33) 31.9 (8.9-77) 3/14 10/14 8/14
0.221 (0.13-1.4) 0.156 (0.06-0.44) 0.577 (0.16-1.9) 12.1 (8.6-13.8) 220 (169-479) 14/14
3.5
3/14, poor responses
8 (4.2-15) 0.64 (0.24-1.7) 0.86 (0.1-2.5)
0.37 (0.17-0.51) 0.09 (0.04-0.10) 0.02 (0.01-0.05) 0.12 (0.06-0.16)
8/14 5/14 3a
Patients with CXCR2 mutations
4
1.8 (1.2-2.9) 16.3 (7.2-36.5) 4/4
2/4
0/4
0.496 (0.18-0.57) 0.477 (0.29-0.54) 3.2 (1.6-4.5) 11.8 (10.7-12.6) 338 (277-523) 1/4
3
2/4, good responses
16.2 (10.2-20.5) 1.63 (0.81-2.53) 3.33 (1.69-3.96)
0.80 (0.54-1.46) 0.50 (0.29-1.29) 0.38 (0.12-0.77) 0.15 (0.08-0.78)
0/4
0/4
0/4
Relevant differences
** **
* *
** ** **
** ** ** **
Results are expressed as medians (range),unless stated otherwise.Bold type indicates lower values and bold italics indicate higher values compared to the reference range. aTwo deaths occurred between 30 and 40 years of age from vulvar cancer or atypical mycobacteria with liver failure, and one 77-year-old died of pneumonitis. Owing to the very low number of patients to be compared, the most relevant differences are indicated as *P<0.01 or **P<0.001. CBC: complete blood count; G-CSF: granulocyte colony-stimulating factor; NK: natural killer.
(218,988,774_219,002,220) encompassing only CXCR2. To exclude other causal variants in P2, P3 and P4, who harbor missense CXCR2 mutations, DNA from the probands and their parents were subjected to whole- exome sequencing. The mean depth of exome coverage was 74X with 96% covered at least 20X. The CXCR2 mutations were confirmed and no other potentially causative candidate variants were identified. The homozygous CXCR2 genotypes of P1, P2, and P3 were consistent with the reported consanguinity of these pedi- grees. Parents were heterozygous carriers and their blood- cell counts were within normal ranges. The three CXCR2 missense mutations (p.Arg144Cys, p.Arg212Trp and p.Arg289Cys) had been entered into the Genome Aggregation Database (gnomAD) with an allele frequency <5x10–5 but never as being homozygous. The mutation in P2 affects Arg144 which constitutes the critical DRY motif for G-protein activation.7 The mutations affect Arg184 in P3, which is highly conserved between CXCR2 and CXCR1, and Arg212 and Arg289 in P4, which belong to domains cooperating with the CXCR2 N-terminal for the efficient docking of the CXCL8-chemokine ligand (Online Supplementary Figure S1C).8
We then examined cell-surface CXCR2 expression in neutrophils (Figure 1B), monocytes (Online Supplementary Figure S2A) and natural killer cells (data not shown) from P1, P2 and P3, their parents, and healthy control blood donors. As expected, CXCR2 was not expressed in the
different cell populations derived from patient P1, who has a homozygous CXCR2-gene deletion. Her mother, who carries a heterozygous CXCR2 deletion, had inter- mediate CXCR2 expression between P1 and control val- ues. That mutant-dosage effect was also observed in car- riers of CXCR2 missense mutations, e.g., all pedigree-P2 blood cell populations noted above and pedigree-P3 monocytes (Figure 1B, Online Supplementary Figure S2A). Whether the underlying mechanisms implicate altered turnover of the Arg144Cys mutant and, in a more cell- restricted fashion, of the Arg212Trp mutant, remains to be investigated. As expected based on the patients’ WT CXCR4 genotypes, cell-surface CXCR4 expression was within the normal range for all tested blood cell popula- tions as illustrated for P1 and P3 (Online Supplementary Figure S2B).
We evaluated the potential impact of CXCR2 muta- tions on the CXCL8-driven chemotactic response of blood neutrophils derived from P1 and P3 pedigrees (Figure 1C). In transwell migration assays, healthy donors’ neutrophils responded to CXCL8, yielding a typ- ical bell-shaped, dose-dependent, chemotaxis-response curve. Blockade with the specific CXCR2 inhibitor SB265610 confirmed the involvement of CXCR2 in the observed chemotaxis. Neutrophils from parents migrated similarly to controls despite lower cell-surface CXCR2 expression, supporting the reported dissociation between the expression level of chemokines-receptors and their
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