Whim Syndrome Caused By a 5 Base Pair Deletion in the C-Terminus of Chemokine Receptor CXCR4

Introduction: WHIM syndrome is a rare autosomal dominant, primary immunodeficiency disease characterized by warts, hypogammaglobulinemia, infections and myelokathexis (impaired neutrophil egress from bone marrow resulting in neutropenia). Mutations in the C-terminus of chemokine receptor CXCR4 cause almost all known cases of WHIM syndrome (4 truncations, 3 frame-shifts and 1 single amino acid charge-change), and result in impaired ligand-induced receptor internalization and desensitization. This enhances receptor signaling, and exaggerates normal CXCR4 function to retain neutrophils and other leukocytes in bone marrow, resulting in myelokathexis and increased susceptibility to certain infections.

Methods: A 2 year old boy with recurrent infections and neutropenia was referred to the NIH Clinical Center. Four anonymized healthy donors were recruited as homozygous CXCR4^WT controls. Genomic DNA was isolated from peripheral blood and the CXCR4 gene was sequenced using the Sanger method. The eukaryotic expression vector pcDNA3.1(-) was used to make 3 recombinant plasmids encoding the wild type gene, (CXCR4^WT) the most common disease mutation (CXCR4^R334X) and the index patient's mutation (CXCR4^L329fs). K562 cells were transfected with plasmids by nucleofection. Blood leukocyte subsets and CXCR4 expression on PBMCs from healthy donors and the patient and stably transfected K562 cells with or without CXCL12 stimulation were examined by FACS analysis. Calcium flux in response to CXCL12 was analyzed in receptor-transfected and parental cell lines as well as in primary leukocytes.

Results: The boy had recurrent infections, hypogammaglobulinemia, panleukopenia and myelokathexis, consistent with WHIM syndrome but both parents were healthy. Genetic analysis revealed a novel heterozygous mutation in CXCR4, designated CXCR4^L329fs, involving a 5 base pair deletion from nucleotides 986-990 in the open reading frame. This deletion causes a frame-shift at codon 329 that introduces 12 missense amino acids and deletes the conserved C-terminal 24 amino acids. Interestingly a different mutation that causes a frame-shift at the same codon has been recently described as a somatic mutation in one case of Waldenström's macroglobulinemia, which deletes nucleotide 984-986 and inserts a T. The CXCR4^L329fs mutation in our patient is the largest truncation mutant reported to date in WHIM syndrome, and was not identified in unaffected family members or in the general population, suggesting that it is a de novo mutation. As with the most common WHIM mutation, CXCR4^R334X, this new mutation also caused decreased receptor downregulation in response to CXCL12 in both PBMCs from the patient as well as in K562 cells transfected with the cloned receptor and increased CXCR4 calcium flux signaling in stably transfected K562 cells.

Conclusion:CXCR4^L329fs is a novel de novo gain-of-function mutation of the C-terminus of CXCR4 that causes WHIM syndrome.