Characterization of a Novel Missense CXCR4 Mutation in a Patient with WHIM-like Syndrome

Background: WHIM (Warts, Hypogammaglobulinemia, Infections, Myelokathexis) syndrome is a rare primary immunodeficiency with a heterogeneous presentation of symptoms defining its acronym as well as panleukopenia. The majority of cases are inherited in an autosomal dominant manner, with gain-of-function mutations in the C-terminus of the C-X-C chemokine receptor 4 (CXCR4) (McDermott D, et al. Immunol Rev. 2019;287:91-102). To our knowledge, there are no literature reports implicating CXCR4^WHIM mutations outside the CXCR4 C-terminus. Here we report the clinical presentation of a patient with the novel mutation CXCR4^D84H and characterize the functional effects of the mutation on CXCR4 trafficking and chemotaxis in in vitro and ex vivo assays. Further, CXCR4^D84H is reported in 3 population genetic databases (average allele frequency 3.8 × 10 ^-5) and is also found in 5 unrelated patients in another rare disease database (CentoMD).

C ase Report: A 40-year-old female presented with a history of recurrent vulvovaginal and anal dysplasia and carcinoma in situ requiring multiple surgeries starting at age 20 years. Cytopenia was first documented at age 15 years in the context of mononucleosis, which resolved, although cytopenias persisted through adulthood. To date, the patient has decreased absolute neutrophil counts (600-1100/mm ³ range), WBC counts ~2000/mm ³, and normal immunoglobulin levels. Bone marrow biopsy revealed bilobed neutrophils and granulocyte precursors. The patient has no history of recurrent infections besides HPV/EBV and no family history of warts, immunodeficiency, or squamous cell carcinoma. Genetic testing revealed a heterozygous mutation in CXCR4 (c.250G>C, D84H) that has not been previously reported. This missense mutation occurs in the transmembrane domain of CXCR4 in proximity of the residues involved in signal initiation, and hence, may alter signaling responses.

Methods: Peripheral blood mononuclear cells (PBMCs) were isolated from this patient and 2 healthy controls and analyzed for functionality ex vivo including CXCR4 internalization assays and chemotaxis to exogenously added CXCR4-ligand CXCL12. The CXCR4-negative K562 cell line was transiently transfected to express CXCR4^D84H, CXCR4^WT, and known CXCR4^WHIM mutations for further comparison of their functional responses to CXCL12.

Results: Flow cytometry data shows abnormal peripheral leukocyte frequency and count. Lymphocyte subpopulations showed 33%-57% reduced CXCR4 internalization in response to CXCL12 compared to controls. Chemotactic response to CXCL12 was increased in PBMC subsets including natural killer T (NKT) cells compared to healthy donor cells. These observations are comparable to previously published responses to pathogenic CXCR4^WHIM variants, but, chemotactic responses of NKT cells is a novel finding. In K562-transfected cells, CXCR4^D84H demonstrated impaired CXCL12-induced internalization compared to CXCR4^WT at a level comparable to the CXCR4^E343K variant, the only CXCR4 missense variant known to date to cause WHIM syndrome. Chemotaxis of cells expressing CXCR4^D84H in response to CXCL12 is increased by 1.25- to 2.4-fold compared to CXCR4^WTdepending on CXCL12 concentration, in line with the observation of increased chemotaxis of cells expressing known CXCR4^WHIM variants. Overall, the CXCR4^D84H mutation recapitulates the phenotypes exhibited by known CXCR4^WHIM variants in both internalization and chemotaxis assays.

Conclusions: We show the functional effects of a novel missense mutation CXCR4^D84H found in a patient with clinical WHIM syndrome recapitulates the internalization and chemotaxis findings of other CXCR4^WHIM mutations. This is the first report of a missense mutation in CXCR4 outside of the C-terminus causing a WHIM phenotype, and additionally highlights defective chemotaxis in NKT cells, which may be relevant in this patient's HPV-associated carcinoma in situ phenotype. Based on an analysis of population databases, and assuming a conservative 5-10% penetrance, there are potentially ~1250-2500 individuals in the United States with disease due to the p.D84H variant alone, highlighting the under-recognition of WHIM syndrome. Patients who have similar clinical presentation as described in this case study should be evaluated for WHIM syndrome and other primary immunodeficiencies by using genetic screening for CXCR4 and diagnostic assays described.