Common variable immunodeficiency and natural killer cell lymphopenia caused by Ets-binding site mutation in the IL-2 receptor γ (IL2RG) gene promoter.

To the Editor:

Patients with severe combined immunodeficiency (SCID) of a classical phenotype present within the first year of life with life-threatening infections and failure to thrive. The X-linked T−B+ natural killer (NK)− form of SCID is the most frequent type (44% to 46%) and is a consequence of mutations in the IL-2 receptor γ (IL2RG) gene (OMIM 308380), which encodes the common cytokine receptor γ chain (γc). The γc acts as a signal-transducing subunit of cytokine receptors that are essential in the ontogeny and function of T, B, and NK cells, namely IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21. The intracellular part of γc interacts with Janus kinase 3 and mediates phosphorylation and activation of signal transducer and activator of transcription (STAT) proteins, which regulate induction of gene transcription.

A number of patients with a milder form of combined immunodeficiency, often termed “leaky” or “hypomorphic” SCID, have been described. Here we describe 2 male relatives with a novel hypomorphic mutation in the IL2RG promoter who presented with a phenotype more akin to common variable immunodeficiency (CVID). CVID is the most common clinically and genetically heterogeneous primary immunodeficiency, which is characterized by low IgG, IgA, and/or IgM levels, with a failure to produce specific antibodies. Mutations in genes encoding transmembrane activator and CAML interactor (TACI), inducible costimulator (ICOS), CD19, CD20, CD21, CD81, LRBA, CXCR4, NF-κB2, B cell–activating factor of the TNF family (BAFF) receptor, TNF-related weak inducer of apoptosis (TWEAK), phosphoinositide 3-kinase catalytic subunit δ polypeptide (PI3KCD), and PI3KR1 were shown to cause CVID-like phenotypes.

The grandson presented at age 4 years with a history of recurrent bacterial otitis media and chronic suppurative rhinitis, rotavirus-induced gastroenteritis (age 18 months), echoviral gastroenteritis (age 2 years), and varicella zoster (age 4 years). He had IgG deficiency (1.8 g/L) with normal IgA and IgM levels (1.0 and 0.5 g/L, respectively) and did not mount an adequate response to the 23-valent pneumococcal polysaccharide vaccine (Pneumovax; Merck & Co, Whitehouse Station, NJ), although he responded appropriately to immunization with protein antigens (see Table E1 in this article's Online Repository at www.jacionline.org). He had normal numbers of T and B cells but completely absent NK cells. T-cell proliferation after stimulation with PHA, anti-CD3, and Candida species was suboptimal but not completely abrogated. He was started on immunoglobulin replacement therapy and is well, with his infections limited to recalcitrant cutaneous warts.

Table E1

Summary of immunologic investigations in the patients

Investigation	Grandson 44 y (normal range)	Grandson 4 y (normal range)
CD3+ cells (109/L)	0.83 (0.7-2.1)	4.34 (0.9-4.5)
CD19+ cells (109/L)	0.01 (0.1-0.5)	1.10 (0.2-2.1)
CD16+CD56+ cells (109/L)	0.00 (0.09-0.6)	0.00 (0.1-1.0)
CD3+CD4+ cells (109/L)	0.25 (0.3-1.4)	2.86 (0.5-2.4)
CD3+CD8+ cells (109/L)	0.53 (0.2-0.9)	1.21 (0.3-1.6)
γδ T cells (109/L)	0.03	1.39
Naive CD4+ T cells (CD27+CD45RA+)		50% (>50%)
Naive CD8+ T cells (CD27+CD45RA+)		61% (>50%)
PHA, 0 μg/mL (mean CPM)	68	57
PHA, 4 μg/ml (mean CPM)	3,859 (>12,000)	10,909 (>12,000)
CD3 background (mean CPM)	167	183
CD3 stimulated (mean CPM)	484 (>7,500)	3,952 (>7,500)
Candida species background (mean CPM)	630	904
Candida species stimulated (mean CPM)	5,274 (>12,500)	9,252 (>12,500)
IgG (g/L)	7.2 (6-13)	1.8 (4.9-16.1)
IgG1 (g/L)	4.66 (3.1-8.9)
IgG2 (g/L)	0.58 (1.4-5.5)
IgG3 (g/L)	0.26 (0.04-1.07)
IgG4 (g/L)	0.00 (0.01- 0.93)
IgA (g/L)	2.3 (0.8-3.7)	1.0 (0.4-2.0)
IgM (g/L)	2.4 (0.4-2.2)	0.5 (0.5-2.0)
CD4+ TRECs per million T cells		19,057 (>20,000)
CD8+ TRECs per million T cells		34,125 (>20,000)
Naive B cells (IgD+IgM+CD27−)		98% (70% to 90%)
Anti-tetanus antibody (IU/mL)	<10 IU (undetectable)	0.28 (protective range)
Anti-pneumococcal antibody	<10 IU (undetectable); no response on immunization	220 before immunization/352 after immunization (640 units of range for unimmunized subject)
Anti-rubella antibody		4 (>10)
Anti–varicella zoster antibody		43 (>20)
Anti-mumps antibody		IgG positive
Anti-measles antibody		1.4 (detectable)
Anti-HiB antibody (μg/mL)		2.17 before immunization/>14 after immunization (optimal >1)

CPM, Counts per minute; TRECs, T-cell receptor excision circles.

At the time of his diagnosis, it was noted that his maternal grandfather was under treatment for CVID. The grandfather presented to an immunology team at the age of 34 years with a 20-year history of recurrent otosinopulmonary tract infections with Streptococcus pneumoniae and Haemophilus influenzae, bronchiectasis, and type 1 diabetes mellitus and celiac disease. On initial presentation, he had an IgG2 and IgG4 subclass deficiency, absent antibody response to polysaccharide vaccine, CD4 and NK lymphopenia, and reduced proliferative responses to PHA. Immunoglobulin substitution was implemented, along with antibiotic prophylaxis, and he was managed successfully on this regimen for 25 years until he died at age 62 years after a cardiac event.

Flow cytometric analysis of lymphocytes revealed a significantly diminished γc expression in both the grandson and grandfather (Fig 1, A; see the Methods section in this article's Online Repository at www.jacionline.org). Likewise, IL2RG mRNA expression in sorted T and B cells from the grandson showed a 4.2-fold reduction in T cells and a 33-fold reduction in B cells compared with healthy control subjects (Fig 1, B). The T cells were then stimulated with IL-2, IL-7, and IL-15, and phosphorylated STAT5 levels were determined by means of flow cytometric analysis (Fig 1, C). This was diminished in the patient with a fold reduction compared with healthy control samples of 3.5-, 7.5-, and 3.8-fold for IL-2, IL-7, and IL-15, respectively.

Fig 1

Reduced IL2RG expression and function. A and B, Common γc expression on lymphocytes (Fig 1, A) in a control subject (CON), the grandson (GS), and the grandfather (GF), as well as IL2RG mRNA expression (Fig 1, B). C, Phosphorylated STAT5 (pSTAT5) expression after stimulation with cytokines (dark gray) or unstimulated (light gray). Mean fluorescence intensities are shown in parentheses. D, Illustration of the point mutation in the IL2RG promoter with the ETS consensus sequence underscored. E, Expression of common γc after transduction of ED7R cells. The vector copy number (VCN) per cell is shown.

X-inactivation studies performed on samples from the mother of the grandson demonstrated random X-inactivation in whole blood but apparent nonrandom X-inactivation in T cells (see Table E2 in this article's Online Repository at www.jacionline.org). Whole-exome sequencing of the grandson revealed a point mutation, C to T at position g.chrX:71,111,618 (GRCh38), which was located −13 nucleotides upstream of the transcription start site in the IL2RG gene (ENST00000374202; Fig 1, D). This is situated at an identified binding site for the transcription factor ETS, which is required for basal promoter activity in cell lines.

Table E2

X-inactivation studies of the 3 generations in the family

Relationship	AR (CAG)n−HpaII	AR (CAG)n+HpaII	Conclusion
Son (grandson)	272	NA	High-risk X-chromosome
Mother of grandson	Whole blood 272, 292T cells 272, 292	272, 292272	Nonrandom X- inactivation in T cells
Grandfather	272		High-risk X-chromosome

For functional validation, we generated the same mutation in an IL2RG minigene (Mut.gcPRO and WT.gcPRO), and using a lentiviral vector, introduced this into γc-deficient ED7R cells. We found that γc expression from Mut.gcPRO was dramatically abrogated when compared with the wild-type sequence in a dose-dependent manner. When transduced at similar efficiency (similar vector copy numbers), there is an 8-fold difference in γc expression between the WT-gcPRO and Mut-gcPRO transduced cells (Fig 1, E). To confirm that the mutation abrogated binding of ETS, using the electrophoretic mobility shift assay, we showed that mutant oligonucleotides were unable to form a normal protein/DNA complex (Fig 2).

Fig 2

Electrophoretic mobility shift assay. Biotin-labeled wild-type or mutant oligonucleotides incubated without nuclear extracts (lanes 1 and 4), with nuclear extracts (lanes 2, 3, 5, and 6), and in the absence (lanes 2 and 5) or presence (lanes 3 and 6) of an excess of unlabeled oligonucleotides. A supershift DNA/protein complex band is detected and marked. The free-labeled oligonucleotide is indicated.

More than 100 mutations in IL2RG have been described extending across all of its 8 exons, intron/exon boundaries, and 3′ regulatory regions. Although most of the known mutations result in a classical immunophenotype of T−B+NK− SCID, variants leading to a T−B+NK+ SCID and TlowB+NK+ have been described.5, 6 Attenuated SCID phenotypes have also been observed as a result of splice-site mutations resulting in diminished expression of truncated γc protein or as a result of somatic reversion.5, 6, 7

Here we identified a novel point mutation at nucleotide −13 upstream of the transcription start site in a putative ETS-binding site. ETS transcription factors comprise a large evolutionarily conserved family characterized by sequence homology within their DNA-binding domain that bind to sequences containing a consensus GGAA/T motif. The ETS transcription factors have been linked with diverse biological processes, including hematopoiesis, T-cell survival, and NK cell production. Previous studies have shown that an ETS-binding site in a 1053-bp fragment 5′ to the IL2RG transcription initiation site is essential for tissue-specific basal promoter activity of IL2RG.

Our data indicate that a point mutation within the ETS-binding site of the proximal IL2RG promoter has a significant detrimental effect on its activity in human subjects. The residual expression of γc appears to differentially affect signaling through the cytokine receptors leading to normal T-cell development, with minimal reduction in T-cell function and absent NK cell development. In this family this resulted in an initial presentation akin to CVID, manifesting with recurrent bacterial and viral infections. This scenario should be considered in male patients with antibody deficiency, particularly if accompanied by NK lymphopenia. These patients should also be monitored closely for more serious manifestations because this defect is amenable to correction by means of hematopoietic stem cell transplantation or gene therapy. Furthermore, our finding highlights the potential role of mutations in gene regulatory regions as a cause of significant primary immunodeficiencies.