In three families with a dominantly inherited syndrome of cold urticaria and pleiotropic immune dysregulation, we found that genomic deletions in PLCG2 were responsible for this unique phenotype. Diminished receptor-mediated signaling in PLAID B cells resulted in abnormal activation, class-switch recombination, and receptor editing, which have been associated with antibody deficiency, recurrent infection, and impaired central tolerance. Furthermore, the signaling defect in PLAID was temperature-sensitive, as shown by the enhanced signaling and cellular activation observed at subphysiologic temperatures, even in the absence of surface-receptor ligation in B cells and mast cells expressing mutant PLCγ2.
The PLAID-associated PLCG2
deletions affected the regulatory region, including the autoinhibitory cSH2 domain, which normally couples the enzymatic activity of PLCγ2
to upstream pathways.21
PLAID-associated deletions resulted in the failure of autoinhibition and constitutive phospholipase activity, as measured by standard enzymatic assays. These data are consistent with the hypothesis that, at rest, cSH2 obstructs the PLCγ2
catalytic site and that this inhibitory interaction is relieved by phosphorylation of critical tyrosine residues, which facilitates a conformational change in PLCγ2
, thereby exposing the catalytic site.21-23
Nonetheless, B cells and natural killer cells from subjects with PLAID clearly had decreased PLCγ2
-dependent signaling and function, with responses similar to those observed in Plcg2-
This apparently paradoxical loss of downstream function may be the direct result of chronic signaling, just as chronic B-cell–receptor stimulation results in calcium currents of diminished amplitude, alteration of signaling cascades, and ultimately, proliferative anergy.24-26
Although the specific mechanisms that underlie the reduction in PLCγ2
-mediated signal transduction remain to be elucidated, there are several possible explanations. Increased phospholipase activity may lead to depletion of the substrate PIP2
proximate to PLCγ2
, resulting in impaired IP3
production and IP3
-mediated calcium release.27
Alternatively, excessively high concentrations of the products of PLCγ2
may induce feedback-mediated down-regulation of the distal signaling pathways.28
Both these mechanisms suggest that phospholipase inhibitors29
might be used to treat PLAID. It is also possible that the deleted regions have other functions that are critical for PLCγ2
-mediated signaling, in addition to the phospholipase activity.30
Cold urticaria in patients with PLAID is characterized by mast-cell degranulation after cold stimulation, which is similar to the lesions reported in other types of cold urticaria.7,9
Previous serum transfer experiments have suggested that causative autoantibodies may underlie some cases of cold urticaria.9,31
However, in our study, B cells and mast cells expressing the mutant forms of PLCγ2
had increased cellular activation and effector function at subphysiologic temperatures without receptor stimulation, indicating that the altered function of mutant PLCγ2
was responsible for cold urticaria in the subjects in a dominant, cell-intrinsic fashion.
There are two gain-of-function Plcg2
mouse models, but they differ from PLAID in that these mice lack constitutively activated PLCγ2
. In addition, the mouse cells are subject to abnormally high calcium flux after surface-receptor ligation, and all the mice have severe inflammatory arthritis.32,33
Furthermore, the autoimmunity in subjects with PLAID and in one of these mouse models (Plcg2Ali5
mice) appears to develop through different mechanisms, given that the induction of intracellular signaling by receptor stimulation is reduced in PLAID B cells but increased in Plcg2Ali5
B cells. Although cold urticaria is not manifested in either of these mouse models, Plcg2Ali5
mice have inflammatory dermatitis on the coolest body parts (the ears and distal extremities), resembling the distribution of cutaneous granulomatous lesions in several subjects with PLAID in our study.
Common variable immunodeficiency (CVID) is a prototypic antibody-deficiency disorder, which is also associated with autoimmunity. Although CVID was present in only three subjects in our study, there is substantial phenotypic overlap between PLAID and CVID. In addition to antibody deficiency and autoimmunity, common features of the two disorders include granulomatous disease,12
diminished class-switched memory B cells,17
impaired B-cell calcium flux,34
and low numbers of natural killer cells.35
Like PLAID, CVID is associated with mutations that affect proteins involved in B-cell activation.36-40
Given the overlap of pathways and phenotypes between the two diseases, it seems likely that an understanding of PLAID will provide mechanistic insights into the pathogenesis of CVID and CVID-associated symptoms.
The deletions that we discovered in the three families occur in a region of PLCG2 that is rich in repetitive elements known to facilitate aberrant recombination events. Indeed, five of the six deletion breakpoints occurred within repetitive elements, suggesting that sporadic cases of PLAID may be caused by similar de novo or even somatic mutations in PLCG2.
PLAID-associated PLCG2 mutations and the resulting phenotypes provide a rare example of a monogenic disease defined by an atopic phenotype. The effects of the mutant forms of PLCγ2 in this complex disorder illuminate vital elements of phospholipase-mediated signaling, including its role in leukocyte function, host defense, and self-tolerance. In addition, such effects illuminate how novel genetic variants can cause atypical temperature responses by altering the balance among complex biologic pathways.