A hypermorphic missense mutation in PLCG2, encoding phospholipase Cγ2, causes a dominantly inherited autoinflammatory disease with immunodeficiency.

Whole-exome sequencing was performed in a family affected by dominantly inherited inflammatory disease characterized by recurrent blistering skin lesions, bronchiolitis, arthralgia, ocular inflammation, enterocolitis, absence of autoantibodies, and mild immunodeficiency. Exome data from three samples, including the affected father and daughter and unaffected mother, were filtered for the exclusion of reported variants, along with benign variants, as determined by PolyPhen-2. A total of eight transcripts were identified as possible candidate genes. We confirmed a variant, c.2120C>A (p.Ser707Tyr), within PLCG2 as the only de novo variant that was present in two affected family members and not present in four unaffected members. PLCG2 encodes phospholipase Cγ2 (PLCγ2), an enzyme with a critical regulatory role in various immune and inflammatory pathways. The p.Ser707Tyr substitution is located in an autoinhibitory SH2 domain that is crucial for PLCγ2 activation. Overexpression of the altered p.Ser707Tyr protein and ex vivo experiments using affected individuals' leukocytes showed clearly enhanced PLCγ2 activity, suggesting increased intracellular signaling in the PLCγ2-mediated pathway. Recently, our laboratory identified in individuals with cold-induced urticaria and immune dysregulation PLCG2 exon-skipping mutations resulting in protein products with constitutive phospholipase activity but with reduced intracellular signaling at physiological temperatures. In contrast, the p.Ser707Tyr substitution in PLCγ2 causes a distinct inflammatory phenotype that is not provoked by cold temperatures and that has different end-organ involvement and increased intracellular signaling at physiological temperatures. Our results highlight the utility of exome-sequencing technology in finding causal mutations in nuclear families with dominantly inherited traits otherwise intractable by linkage analysis.

Cold urticaria, immunodeficiency, and autoimmunity related to PLCG2 deletions.

BACKGROUND: Mendelian analysis of disorders of immune regulation can provide insight into molecular pathways associated with host defense and immune tolerance. METHODS: We identified three families with a dominantly inherited complex of cold-induced urticaria, antibody deficiency, and susceptibility to infection and autoimmunity. Immunophenotyping methods included flow cytometry, analysis of serum immunoglobulins and autoantibodies, lymphocyte stimulation, and enzymatic assays. Genetic studies included linkage analysis, targeted Sanger sequencing, and next-generation whole-genome sequencing. RESULTS: Cold urticaria occurred in all affected subjects. Other, variable manifestations included atopy, granulomatous rash, autoimmune thyroiditis, the presence of antinuclear antibodies, sinopulmonary infections, and common variable immunodeficiency. Levels of serum IgM and IgA and circulating natural killer cells and class-switched memory B cells were reduced. Linkage analysis showed a 7-Mb candidate interval on chromosome 16q in one family, overlapping by 3.5 Mb a disease-associated haplotype in a smaller family. This interval includes PLCG2, encoding phospholipase Cγ(2) (PLCγ(2)), a signaling molecule expressed in B cells, natural killer cells, and mast cells. Sequencing of complementary DNA revealed heterozygous transcripts lacking exon 19 in two families and lacking exons 20 through 22 in a third family. Genomic sequencing identified three distinct in-frame deletions that cosegregated with disease. These deletions, located within a region encoding an autoinhibitory domain, result in protein products with constitutive phospholipase activity. PLCG2-expressing cells had diminished cellular signaling at 37°C but enhanced signaling at subphysiologic temperatures. CONCLUSIONS: Genomic deletions in PLCG2 cause gain of PLCγ(2) function, leading to signaling abnormalities in multiple leukocyte subsets and a phenotype encompassing both excessive and deficient immune function. (Funded by the National Institutes of Health Intramural Research Programs and others.).

Lymphocyte proliferation in immune-mediated diseases.

Defects in T cell homeostatic mechanisms can result in T cell lymphopenia, defined as decreased numbers of lymphocytes. Lymphopenia results in homeostatic proliferation in order to maintain T cell homeostasis. It has been proposed that homeostatic proliferation can expand the pool of autoreactive T cells that promote autoimmunity, and indeed recent studies have further substantiated this observation in both animal models and humans. Conversely, homeostatic proliferation can promote tumor immunity by allowing tumor-specific T cells to accumulate. In this review, we discuss how the outcome of homeostatic proliferation can function both in a deleterious manner in autoimmunity and a beneficial way in tumor immunity. We also discuss the roles of various cytokines and T regulatory cells that control homeostatic proliferation.

Spatial distribution and function of sterol regulatory element-binding protein 1a and 2 homo- and heterodimers by in vivo two-photon imaging and spectroscopy fluorescence resonance energy transfer.

Sterol regulatory element-binding proteins (SREBPs) are a subfamily of basic helix-loop-helix-leucine zipper proteins that regulate lipid metabolism. We show novel evidence of the in vivo occurrence and subnuclear spatial localization of both exogenously expressed SREBP-1a and -2 homodimers and heterodimers obtained by two-photon imaging and spectroscopy fluorescence resonance energy transfer. SREBP-1a homodimers localize diffusely in the nucleus, whereas SREBP-2 homodimers and the SREBP-1a/SREBP-2 heterodimer localize predominantly to nuclear speckles or foci, with some cells showing a diffuse pattern. We also used tethered SREBP dimers to demonstrate that both homo- and heterodimeric SREBPs activate transcription in vivo. Ultrastructural analysis revealed that the punctate foci containing SREBP-2 are electron-dense nuclear bodies, similar or identical to structures containing the promyelocyte (PML) protein. Immunofluorescence studies suggest that a dynamic interplay exists between PML, as well as another component of the PML-containing nuclear body, SUMO-1, and SREBP-2 within these nuclear structures. These findings provide new insight into the overall process of transcriptional activation mediated by the SREBP family.

Activation domains from both monomers contribute to transcriptional stimulation by sterol regulatory element-binding protein dimers.

Sterol regulatory element-binding proteins (SREBPs) are basic helix-loop-helix leucine zipper proteins that act as dimers to activate genes in lipid metabolism. Three SREBP isoforms, 1a, 1c, and 2, are expressed at varying levels in different tissues. Thus, homo- and heterodimers probably contribute to overall SREBP activity. No studies have directly evaluated the formation or activation properties of SREBP homo- and heterodimers. Studies with overexpressed SREBP monomers are inconclusive regarding the function of a particular SREBP dimer because of potential dimerization with endogenous proteins. To assess activation by a particular SREBP dimer, we fused DNA encoding individual monomers together via a predicted flexible polypeptide tether. Tethered SREBP dimers bound DNA equivalently to the monomeric proteins and were resistant to dominant negative SREBP-1 inhibition, confirming preferential formation of intramolecular dimers. Tethered SREBP-1a and -2 homodimers, similar to the monomeric forms, activated target genes more robustly than tethered SREBP-1c homodimers. A forced SREBP-1a/2 heterodimer had similar activity to the respective homodimers. However, SREBP-1c in a heterodimer with either SREBP-1a or -2 attenuated the activity relative to the SREBP-1a or -2 homodimers. These experiments provide some of the first data showing that the integrity of both activation domains in a dimeric transcription factor is required for maximal activity. In addition, the results support a model where changes in SREBP-1c protein expression that occur in response to insulin signaling and liver X receptor signaling would be predicted to increase or decrease overall SREBP activity in a tissue-specific fashion depending on the initial fractional contribution of SREBP-1c to total cellular levels of SREBP.