WHIM syndrome is a rare IEI, which was first described in 1964 [22]. The acronym WHIM was coined in 1990 to represent the four main clinical manifestations: warts, hypogammaglobulinemia, recurrent infection, and myelokathexis [1]. However, an association between heterozygous mutations in the chemokine receptor CXCR4 gene and this syndrome was found until 2003 [4]. Here, we report a novel extended mutation caused by a pair of base duplications (c.1016_1017dupCT, p. V340L fs*27) in the C-terminal region of CXCR4, which resulted in recurrent infections and aperiodic neutropenia in two generations of a Chinese family. P4 had skin warts on fingers, and P1 and P3 had myelokathexis, but none of the three patients had hypogammaglobulinemia.
The clinical manifestations of WHIM syndrome are extremely heterogeneous [10]; indeed, only about one third of patients fulfill the diagnostic tetrad of WHIM syndrome. Most patients have non-cyclic neutropenia (98%), followed by lymphopenia (88%) and hypogammaglobulinemia (65%). Recurrent infections occur in 92% of patients, mainly otitis media and pneumonia, and 40% of patients have HPV-related warts on different body parts [9]. None of the patients in family 1 had all four features of WHIM syndrome; by contrast, P5 from family 2 presented with warts, hypogammaglobulinemia, infection, and myelokathexis, as well as bronchiectasis and conductive deafness caused by recurrent infections. Since the clinical manifestations of the patients in family 1 were milder than those of P5, we searched the literature for patients with CXCR4 mutations similar to family 1. A previous study reported that a truncated mutation (S339fsX342) caused by a c.1016_1017 deletion at the C-terminus results in all four features of WHIM syndrome; the patient also had persistent enteritis and was treated with G-CSF, subcutaneous immunoglobulin, and antibiotics. The clinical manifestations of this WHIM patient with the extended mutation (S341fsX365) were very similar to those of P1–P4, including warts, neutropenia, recurrent respiratory infections, and myelokathexis, but no hypogammaglobulinemia. Moreover, the patient only received IVIG treatment for 3 years when he was young [23]. A recently reported case of WHIM (S339L fs*27) presented with myelokathexis and recurrent infections, without warts or hypogammaglobulinemia [9]. Therefore, the relationship between the clinical phenotype of WHIM syndrome and mutations needs to be further verified.
Previous studies show that the CXCR4 C-tail harbors multiple potential phosphorylation sites (e.g., Ser324/5, Ser330, Ser339 and Ser346/7), which are rapidly phosphorylated following activation by CXCL12 [21, 24, 25]. It is believed that CXCR4 mutations result in loss of some phosphorylation sites in the C-tail, which hinders β-arrestin recruitment and receptor degradation, and prolongs the residence time of CXCR4 on the cell surface. After binding to its ligand (CXCL12), downstream signals are activated continuously, resulting in the abnormal retention and accelerated apoptosis of mature neutrophils in the bone marrow [26–28]. We found that CXCL12-mediated receptor internalization was markedly impaired in PBMCs harboring the CXCR4V340fs mutation. When HEK293T cells were transfected with the CXCR4WT, CXCR4V340fs, and CXCR4R334X plasmids, the CXCR4V340fs and CXCR4R334X mutation resulted in enhanced and prolonged p-AKT and p-ERK1/2 activation upon CXCL12 stimulation, which is consistent with the effects of gain-of-function mutations associated with WHIM syndrome.
Lymphocytes are generated from hematopoietic stem cells in the bone marrow. The CXCR4/CXCL12 axis is essential for bone marrow colonization during ontogeny, as well as for hematopoietic stem cell homeostasis [29]. Meanwhile, CXCR4 plays an important role in coordinating innate and adaptive immune responses by regulating leukocyte transport and distribution from peripheral tissues, and by maintaining T cell priming by contributing immune synapse formation and stability [30, 31]. Circulating T cell numbers may be reduced in WHIM patients, as previously reported, but not as severely as those of B cells and neutrophils. CD4+T cell numbers may be normal, or only slightly reduced, while CD8+T numbers appear to be affected more. Moreover, naïve T cell subset numbers are reduced, which may be due to reduced thymic output [17, 32, 33]. We also found that TRECs decreased in P2–P4. Furthermore, naïve T cell, especially naïve CD8+T cell, numbers in the peripheral blood of the patients were significantly reduced. Whether this result was associated with decreased expression of CXCR4 on the surface of CD8+T cells rather than CD4+T cells requires further research.
CXCR4 is crucial for the homing, development, and function of B cells. At all stages of development, B cells normally express CXCR4 (at markedly higher levels than most other leukocyte subsets) [6, 34]. Targeted deletion of CXCR4 from B cells leads to premature egress of B cell precursors from the bone marrow, and localization within splenic follicles, resulting in a reduction in the number of mature B cells in the splenic marginal zone and primary follicles, and in defective T-independent antibody responses [35, 36]. WHIM patients show a reduced capacity to generate hypermutated IgG, and an inability to mount antigen-specific memory responses [34]. Here, we found that CXCR4 expression on the surface of B cells from all patients was significantly lower than that on those from HCs. The number of B cell subsets were reduced markedly in our patients. Notably, P5 had the lowest number of B cell subsets. KRECs was also decreased in P2–P4. Furthermore, the amount of PT-specific IgG produced by memory B cells from P2 was also reduced. Although a reduction in B cells is a prominent hematological phenotype of WHIM patients [37], it is unclear how patients with severe B lymphopenia avoid hypogammaglobulinemia.
WHIM syndrome is a relatively benign IEI with low mortality rates. Currently, there is no standardized treatment; the main aim is to prevent HPV-related malignant tumors and chronic sequelae due to recurrent infections (e.g., bronchiectasis and hearing loss). It is generally believed that regular IVIG infusion and prophylactic antibiotics can prevent recurrent lung-ear infections. G-CSF can effectively mobilize neutrophils from the bone marrow to the blood [10, 38]. A clinical phase III trial of the CXCR4 antagonist plerixafor has been completed; the results confirmed that it is better than G-CSF with respect to wart regression and hematologic improvement [39]. Although the recurrent infections in P1 and P2 improved after irregular IVIG treatment (P3 and P4 did not receive treatment), we still recommend long-term and regular IVIG infusion to prevent organ damage.
In conclusion, we identified a novel, frameshift, extended, gain-of-function mutation in CXCR4 that causes familial WHIM syndrome. As in other reported patients, the CXCR4V340fs mutation results in impaired ligand-induced receptor downregulation, as well as increased downstream signaling. However, patients with the CXCR4V340fs mutation have milder clinical symptoms and a less severe immunological phenotype than those with the CXCR4R334X mutation. These results provide new insight into the clinical and immunological features of WHIM syndrome.