Combined immunodeficiency with life-threatening EBV-associated lymphoproliferative disorder in patients lacking functional CD27

Abstract

CD27, a tumor necrosis factor receptor family member, interacts with CD70 and influences T-, B- and NK-cell functions. Disturbance of this axis impairs immunity and memory generation against viruses including Epstein Barr virus (EBV), influenza, and others. CD27 is commonly used as marker of memory B cells for the classification of B-cell deficiencies including common variable immune deficiency. Flow cytometric immunophenotyping including expression analysis of CD27 on lymphoid cells was followed by capillary sequencing of CD27 in index patients, their parents, and non-affected siblings. More comprehensive genetic analysis employed single nucleotide polymorphism-based homozygosity mapping and whole exome sequencing. Analysis of exome sequencing data was performed at two centers using slightly different data analysis pipelines, each based on the Genome Analysis ToolKit Best Practice version 3 recommendations. A comprehensive clinical characterization was correlated to genotype. We report the simultaneous confirmation of human CD27 deficiency in 3 independent families (8 patients) due to a homozygous mutation (p. Cys53Tyr) revealed by whole exome sequencing, leading to disruption of an evolutionarily conserved cystein knot motif of the transmembrane receptor. Phenotypes varied from asymptomatic memory B-cell deficiency (n=3) to EBV-associated hemophagocytosis and lymphoproliferative disorder (LPD; n=3) and malignant lymphoma (n=2; +1 after LPD). Following EBV infection, hypogammaglobulinemia developed in at least 3 of the affected individuals, while specific anti-viral and anti-polysaccharide antibodies and EBV-specific T-cell responses were detectable. In severely affected patients, numbers of iNKT cells and NK-cell function were reduced. Two of 8 patients died, 2 others underwent allogeneic hematopoietic stem cell transplantation successfully, and one received anti-CD20 (rituximab) therapy repeatedly. Since homozygosity mapping and exome sequencing did not reveal additional modifying factors, our findings suggest that lack of functional CD27 predisposes towards a combined immunodeficiency associated with potentially fatal EBV-driven hemo-phagocytosis, lymphoproliferation, and lymphoma development.

Figure 1.

Identification of CD27 deficiency in three kindreds. Three pedigrees with CD27 deficiency were identified. (A) Family A was analyzed by homozygosity mapping, revealing a homozygous interval on Chromosome 12. Two siblings of Patient 1 (Family A) had died at nine months and three days of age, respectively, of unknown causes without molecular diagnosis in Turkey; no material for immunological or genetic analyses was available. (B) Flow cytometry showed absent surface expression for CD27 in lymphoid cells including B-cell subsets, shown here in Patient 1 for IgD vs. CD27 staining on CD19-gated B lymphocytes. (C) Exome sequencing was performed in Patient 1 and filtered for homozygous intervals detected exclusively in Patient 1. None of the total of 28 additional single nucleotide or deletions/insertions variants (SNVs or DIVs, respectively) was validated using capillary sequencing, suggesting that the mutation in CD27 alone is causative of the phenotype in Patient 1. (D) Sanger sequencing confirmed the same mutation in CD27 (c. G158A, p. Cys53Tyr) in Patients 1–3 from Family A, Patients 4 and 5 from Family B, and Patients 6–8 from Family C, respectively.